Bispecific t cell activating antigen binding molecules

ABSTRACT

The present invention generally relates to novel bispecific antigen binding molecules for T cell activation and re-direction to specific target cells. In addition, the present invention relates to polynucleotides encoding such bispecific antigen binding molecules, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the bispecific antigen binding molecules of the invention, and to methods of using these bispecific antigen binding molecules in the treatment of disease.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/281,484, filed on Sep. 30, 2016, which claims priority toEuropean Patent Application No. EP 15188035.8, filed Oct. 2, 2015, thedisclosure of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 20, 2020, isnamed 51177-014002_Sequence_Listing_7_20_20_ST25.txt and is 84,348 bytesin size.

FIELD OF THE INVENTION

The present invention generally relates to bispecific antigen bindingmolecules for activating T cells. In addition, the present inventionrelates to polynucleotides encoding such bispecific antigen bindingmolecules, and vectors and host cells comprising such polynucleotides.The invention further relates to methods for producing the bispecificantigen binding molecules of the invention, and to methods of usingthese bispecific antigen binding molecules in the treatment of disease.

BACKGROUND

The selective destruction of an individual cell or a specific cell typeis often desirable in a variety of clinical settings. For example, it isa primary goal of cancer therapy to specifically destroy tumor cells,while leaving healthy cells and tissues intact and undamaged.

An attractive way of achieving this is by inducing an immune responseagainst the tumor, to make immune effector cells such as natural killer(NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumorcells. CTLs constitute the most potent effector cells of the immunesystem, however they cannot be activated by the effector mechanismmediated by the Fc domain of conventional therapeutic antibodies.

In this regard, bispecific antibodies designed to bind with one “arm” toa surface antigen on target cells, and with the second “arm” to anactivating, invariant component of the T cell receptor (TCR) complex,have become of interest in recent years. The simultaneous binding ofsuch an antibody to both of its targets will force a temporaryinteraction between target cell and T cell, causing activation of anycytotoxic T cell and subsequent lysis of the target cell. Hence, theimmune response is re-directed to the target cells and is independent ofpeptide antigen presentation by the target cell or the specificity ofthe T cell as would be relevant for normal MHC-restricted activation ofCTLs. In this context it is crucial that CTLs are only activated when atarget cell is presenting the bispecific antibody to them, i.e. theimmunological synapse is mimicked. Particularly desirable are bispecificantibodies that do not require lymphocyte preconditioning orco-stimulation in order to elicit efficient lysis of target cells.

Several bispecific antibody formats have been developed and theirsuitability for T cell mediated immunotherapy investigated. Out ofthese, the so-called BiTE (bispecific T cell engager) molecules havebeen very well characterized and already shown some promise in theclinic (reviewed in Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260(2011)). BiTEs are tandem scFv molecules wherein two scFv molecules arefused by a flexible linker. Further bispecific formats being evaluatedfor T cell engagement include diabodies (Holliger et al., Prot Eng 9,299-305 (1996)) and derivatives thereof, such as tandem diabodies(Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). A more recentdevelopment are the so-called DART (dual affinity retargeting)molecules, which are based on the diabody format but feature aC-terminal disulfide bridge for additional stabilization (Moore et al.,Blood 117, 4542-51 (2011)). The so-called triomabs, which are wholehybrid mouse/rat IgG molecules and also currently being evaluated inclinical trials, represent a larger sized format (reviewed in Seimetz etal., Cancer Treat Rev 36, 458-467 (2010)).

The variety of formats that are being developed shows the greatpotential attributed to T cell re-direction and activation inimmunotherapy. The task of generating bispecific antibodies suitabletherefor is, however, by no means trivial, but involves a number ofchallenges that have to be met related to efficacy, toxicity,applicability and produceability of the antibodies.

Small constructs such as, for example, BiTE molecules—while being ableto efficiently crosslink effector and target cells—have a very shortserum half life requiring them to be administered to patients bycontinuous infusion. IgG-like formats on the other hand—while having thegreat benefit of a long half life—suffer from toxicity associated withthe native effector functions inherent to IgG molecules. Theirimmunogenic potential constitutes another unfavorable feature ofIgG-like bispecific antibodies, especially non-human formats, forsuccessful therapeutic development. Finally, a major challenge in thegeneral development of bispecific antibodies has been the production ofbispecific antibody constructs at a clinically sufficient quantity andpurity, due to the mispairing of antibody heavy and light chains ofdifferent specificities upon co-expression, which decreases the yield ofthe correctly assembled construct and results in a number ofnon-functional side products from which the desired bispecific antibodymay be difficult to separate.

Different approaches have been taken to overcome the chain associationissue in bispecific antibodies (see e.g. Klein et al., mAbs 6, 653-663(2012)). For example, the ‘knobs-into-holes’ strategy aims at forcingthe pairing of two different antibody heavy chains by introducingmutations into the CH3 domains to modify the contact interface. On onechain bulky amino acids are replaced by amino acids with short sidechains to create a ‘hole’. Conversely, amino acids with large sidechains are introduced into the other CH3 domain, to create a ‘knob’. Bycoexpressing these two heavy chains (and two identical light chains,which have to be appropriate for both heavy chains), high yields ofheterodimer (‘knob-hole’) versus homodimer (‘hole-hole’ or ‘knob-knob’)are observed (Ridgway, J. B., et al., Protein Eng. 9 (1996) 617-621; andWO 96/027011). The percentage of heterodimer could be further increasedby remodeling the interaction surfaces of the two CH3 domains using aphage display approach and the introduction of a disulfide bridge tostabilize the heterodimers (Merchant, A. M., et al., Nature Biotech. 16(1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35). Newapproaches for the knobs-into-holes technology are described in e.g. inEP 1870459 A1.

The ‘knobs-into-holes’ strategy does, however, not solve the problem ofheavy chain-light chain mispairing, which occurs in bispecificantibodies comprising different light chains for binding to thedifferent target antigens.

A strategy to prevent heavy chain-light chain mispairing is to exchangedomains between the heavy and light chains of one of the binding arms ofa bispecific antibody (see WO 2009/080251, WO 2009/080252, WO2009/080253, WO 2009/080254 and Schaefer, W. et al, PNAS, 108 (2011)11187-11191, which relate to bispecific IgG antibodies with a domaincrossover).

Exchanging the heavy and light chain variable domains VH and VL in oneof the binding arms of the bispecific antibody (WO2009/080252, see alsoSchaefer, W. et al, PNAS, 108 (2011) 11187-11191) clearly reduces theside products caused by the mispairing of a light chain against a firstantigen with the wrong heavy chain against the second antigen (comparedto approaches without such domain exchange). Nevertheless, theseantibody preparations are not completely free of side products. The mainside product is based on a Bence Jones-type interaction (Schaefer, W. etal, PNAS, 108 (2011) 11187-11191; in Fig. S1I of the Supplement). Afurther reduction of such side products is thus desirable to improvee.g. the yield of such bispecific antibodies.

The choice of target antigens and appropriate binders for both the Tcell antigen and the target cell antigen is a further crucial aspect inthe generation of T cell bispecific (TCB) antibodies for therapeuticapplication. Carcinoembryonic antigen (CEA) is an attractive targetantigen as the prevalence of CEA expression is generally high in tumors,but low in normal tissues. Accordingly, numerous antibodies have beenraised against this target, one of which is the murine antibody T84.66(Wagener et al., J Immunol 130, 2308 (1983), Neumaier et al., J Immunol135, 3604 (1985)), which has also been chimerized (WO 1991/01990) andhumanized (WO 2005/086875). WO 2007/071426 or WO 2014/131712 describebispecific antibodies targeting CD3 on T cells and carcinoembryonicantigen (CEA) on target cells.

The present invention provides novel, improved bispecific antigenbinding molecules designed for T cell activation and re-direction,targeting CD3 and CEA, that combine good efficacy and produceabilitywith low toxicity and favorable pharmacokinetic properties.

SUMMARY OF THE INVENTION

The present inventors have developed a novel T cell activatingbispecific antigen binding molecule with unexpected, improved propertiesusing a novel humanized anti-CEA antibody.

Thus, in a first aspect the present invention provides a T cellactivating bispecific antigen binding molecule comprising

(a) a first antigen binding moiety which specifically binds to a firstantigen;(b) a second antigen binding moiety which specifically binds to a secondantigen;wherein the first antigen is an activating T cell antigen and the secondantigen is CEA, or the first antigen is CEA and the second antigen is anactivating T cell antigen; and wherein the antigen binding moiety whichspecifically binds to CEA comprises a heavy chain variable region,particularly a humanized heavy chain variable region, comprising theheavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:14, the HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO: 16, and alight chain variable region, particularly a humanized light chainvariable region, comprising the light chain complementarity determiningregion (LCDR) 1 of SEQ ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and theLCDR 3 of SEQ ID NO: 19.

In one embodiment, the antigen binding moiety which specifically bindsto CEA comprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 22 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 23.

In particular embodiments, the first and/or the second antigen bindingmoiety is a Fab molecule. In a particular embodiment, the second antigenbinding moiety is a Fab molecule which specifically binds to a secondantigen, and wherein the variable domains VL and VH or the constantdomains CL and CH1 of the Fab light chain and the Fab heavy chain arereplaced by each other (i.e. according to such embodiment, the secondFab molecule is a crossover Fab molecule wherein the variable orconstant domains of the Fab light chain and the Fab heavy chain areexchanged).

In particular embodiments, the first (and the third, if any) Fabmolecule is a conventional Fab molecule. In a further particularembodiment, not more than one Fab molecule capable of specific bindingto an activating T cell antigen is present in the T cell activatingbispecific antigen binding molecule (i.e. the T cell activatingbispecific antigen binding molecule provides monovalent binding to theactivating T cell antigen).

In one embodiment, the first antigen is CEA and the second antigen is anactivating T cell antigen.

In a more specific embodiment, the activating T cell antigen is CD3,particularly CD3 epsilon.

In a particular embodiment, the T cell activating bispecific antigenbinding molecule of the invention comprises

(a) a first Fab molecule which specifically binds to a first antigen;(b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH or the constant domains CLand CH1 of the Fab light chain and the Fab heavy chain are replaced byeach other;wherein the first antigen is CEA and the second antigen is an activatingT cell antigen; wherein the first Fab molecule under (a) comprises aheavy chain variable region, particularly a humanized heavy chainvariable region, comprising the heavy chain complementarity determiningregion (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and theHCDR 3 of SEQ ID NO: 16, and a light chain variable region, particularlya humanized light chain variable region, comprising the light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 17, the LCDR 2of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO: 19.

According to a further aspect of the invention, the ratio of a desiredbispecific antibody compared to undesired side products, in particularBence Jones-type side products occurring in bispecific antibodies with aVH/VL domain exchange in one of their binding arms, can be improved bythe introduction of charged amino acids with opposite charges atspecific amino acid positions in the CH1 and CL domains (sometimesreferred to herein as “charge modifications”).

Thus, in some embodiments the first antigen binding moiety under (a) isa first Fab molecule which specifically binds to a first antigen, thesecond antigen binding moiety under (b) is a second Fab molecule whichspecifically binds to a second antigen wherein the variable domains VLand VH of the Fab light chain and the Fab heavy chain are replaced byeach other;

and

-   i) in the constant domain CL of the first Fab molecule under a) the    amino acid at position 124 is substituted independently by lysine    (K), arginine (R) or histidine (H) (numbering according to Kabat),    and wherein in the constant domain CH1 of the first Fab molecule    under a) the amino acid at position 147 or the amino acid at    position 213 is substituted independently by glutamic acid (E), or    aspartic acid (D) (numbering according to Kabat EU index); or-   ii) in the constant domain CL of the second Fab molecule under b)    the amino acid at position 124 is substituted independently by    lysine (K), arginine (R) or histidine (H) (numbering according to    Kabat), and wherein in the constant domain CH1 of the second Fab    molecule under b) the amino acid at position 147 or the amino acid    at position 213 is substituted independently by glutamic acid (E),    or aspartic acid (D) (numbering according to Kabat EU index).

In one such embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 or the amino acid atposition 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In yet another embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In a particular embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) (numbering according to Kabat), and inthe constant domain CH1 of the first Fab molecule under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In another particular embodiment, in the constant domain CL of the firstFab molecule under a) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by arginine (R) (numbering according to Kabat), andin the constant domain CH1 of the first Fab molecule under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In an alternative embodiment, in the constant domain CL of the secondFab molecule under b) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and in the constant domain CH1 of the second Fabmolecule under b) the amino acid at position 147 or the amino acid atposition 213 is substituted independently by glutamic acid (E), oraspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the second Fabmolecule under b) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the second Fabmolecule under b) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In still another embodiment, in the constant domain CL of the second Fabmolecule under b) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and in the constant domain CH1 of the second Fabmolecule under b) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In one embodiment, in the constant domain CL of the second Fab moleculeunder b) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) (numbering according to Kabat), and in theconstant domain CH1 of the second Fab molecule under b) the amino acidat position 147 is substituted by glutamic acid (E) (numbering accordingto Kabat EU index) and the amino acid at position 213 is substituted byglutamic acid (E) (numbering according to Kabat EU index).

In another embodiment, in the constant domain CL of the second Fabmolecule under b) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by arginine (R) (numbering according to Kabat), andin the constant domain CH1 of the second Fab molecule under b) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).

In a particular embodiment, the T cell activating bispecific antigenbinding molecule of the invention comprises

(a) a first Fab molecule which specifically binds to a first antigen;(b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH of the Fab light chain andthe Fab heavy chain are replaced by each other;wherein the first antigen is CEA and the second antigen is an activatingT cell antigen;wherein the first Fab molecule under (a) comprises a heavy chainvariable region, particularly a humanized heavy chain variable region,comprising the heavy chain complementarity determining region (HCDR) 1of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ IDNO: 16, and a light chain variable region, particularly a humanizedlight chain variable region, comprising the light chain complementaritydetermining region (LCDR) 1 of SEQ ID NO: 17, the LCDR 2 of SEQ ID NO:18 and the LCDR 3 of SEQ ID NO: 19; andwherein in the constant domain CL of the first Fab molecule under a) theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat) (in onepreferred embodiment independently by lysine (K) or arginine (R)) andthe amino acid at position 123 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) (inone preferred embodiment independently by lysine (K) or arginine (R)),and in the constant domain CH1 of the first Fab molecule under a) theamino acid at position 147 is substituted independently by glutamic acid(E), or aspartic acid (D) (numbering according to Kabat EU index) andthe amino acid at position 213 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index).

In some embodiments, the T cell activating bispecific antigen bindingmolecule according to the invention further comprises a third antigenbinding moiety which specifically binds to the first antigen. Inparticular embodiments, the third antigen binding moiety is identical tothe first antigen binding moiety. In one embodiment, the third antigenbinding moiety is a Fab molecule.

In particular embodiments, the third and the first antigen bindingmoiety are each a Fab molecule and the third Fab molecule is identicalto the first Fab molecule. In these embodiments, the third Fab moleculethus comprises the same amino acid substitutions, if any, as the firstFab molecule. Like the first Fab molecule, the third Fab moleculeparticularly is a conventional Fab molecule.

If a third antigen binding moiety is present, in a particular embodimentthe first and the third antigen moiety specifically bind to CEA, and thesecond antigen binding moiety specifically binds to an activating T cellantigen, particularly CD3, more particularly CD3 epsilon.

In some embodiments of the T cell activating bispecific antigen bindingmolecule according to the invention the first antigen binding moietyunder a) and the second antigen binding moiety under b) are fused toeach other, optionally via a peptide linker. In particular embodiments,the first and the second antigen binding moiety are each a Fab molecule.In a specific such embodiment, the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule. In an alternative such embodiment, thefirst Fab molecule is fused at the C-terminus of the Fab heavy chain tothe N-terminus of the Fab heavy chain of the second Fab molecule. Inembodiments wherein either (i) the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule or (ii) the first Fab molecule is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the second Fab molecule, additionally the Fab light chainof the Fab molecule and the Fab light chain of the second Fab moleculemay be fused to each other, optionally via a peptide linker.

In particular embodiments, the T cell activating bispecific antigenbinding molecule according to the invention additionally comprises an Fcdomain composed of a first and a second subunit capable of stableassociation.

The T cell activating bispecific antigen binding molecule according tothe invention can have different configurations, i.e. the first, second(and optionally third) antigen binding moiety may be fused to each otherand to the Fc domain in different ways. The components may be fused toeach other directly or, preferably, via one or more suitable peptidelinkers. Where fusion of a Fab molecule is to the N-terminus of asubunit of the Fc domain, it is typically via an immunoglobulin hingeregion.

In one embodiment, the first and the second antigen binding moiety areeach a Fab molecule and the second antigen binding moiety is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the first orthe second subunit of the Fc domain. In such embodiment, the firstantigen binding moiety may be fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second antigenbinding moiety or to the N-terminus of the other one of the subunits ofthe Fc domain.

In one embodiment, the first and the second antigen binding moiety areeach a Fab molecule and the first and the second antigen binding moietyare each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain. In this embodiment,the T cell activating bispecific antigen binding molecule essentiallycomprises an immunoglobulin molecule, wherein in one of the Fab arms theheavy and light chain variable regions VH and VL (or the constantregions CH1 and CL in embodiments wherein no charge modifications asdescribed herein are introduced in CH1 and CL domains) areexchanged/replaced by each other (see FIG. 1A, D).

In alternative embodiments, a third antigen binding moiety, particularlya third Fab molecule, is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first or second subunit of the Fc domain. In aparticular such embodiment, the second and the third antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second Fab molecule. Inthis embodiment, the T cell activating bispecific antigen bindingmolecule essentially comprises an immunoglobulin molecule, wherein inone of the Fab arms the heavy and light chain variable regions VH and VL(or the constant regions CH1 and CL in embodiments wherein no chargemodifications as described herein are introduced in CH1 and CL domains)are exchanged/replaced by each other, and wherein an additional(conventional) Fab molecule is N-terminally fused to said Fab arm (seeFIG. 1B, E). In another such embodiment, the first and the third antigenbinding moiety are each fused at the C-terminus of the Fab heavy chainto the N-terminus of one of the subunits of the Fc domain, and thesecond antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding moiety. In this embodiment, the T cell activatingbispecific antigen binding molecule essentially comprises animmunoglobulin molecule with an additional Fab molecule N-terminallyfused to one of the immunoglobulin Fab arms, wherein in said additionalFab molecule the heavy and light chain variable regions VH and VL (orthe constant regions CH1 and CL in embodiments wherein no chargemodifications as described herein are introduced in CH1 and CL domains)are exchanged/replaced by each other (see FIG. 1C, F).

In a particular embodiment, the immunoglobulin molecule comprised in theT cell activating bispecific antigen binding molecule according to theinvention is an IgG class immunoglobulin. In an even more particularembodiment the immunoglobulin is an IgG₁ subclass immunoglobulin. Inanother embodiment, the immunoglobulin is an IgG₄ subclassimmunoglobulin.

In a particular embodiment, the invention provides a T cell activatingbispecific antigen binding molecule comprising

a) a first Fab molecule which specifically binds to a first antigen;b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH or the constant domains CLand CH1 of the Fab light chain and the Fab heavy chain are replaced byeach other;c) a third Fab molecule which specifically binds to the first antigen;andd) an Fc domain composed of a first and a second subunit capable ofstable association;wherein the first antigen is CEA and the second antigen is an activatingT cell antigen, particularly CD3, more particularly CD3 epsilon;wherein the third Fab molecule under c) is identical to the first Fabmolecule under a); wherein(i) the first Fab molecule under a) is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the secondFab molecule under b), and the second Fab molecule under b) and thethird Fab molecule under c) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder d), or(ii) the second Fab molecule under b) is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the firstFab molecule under a), and the first Fab molecule under a) and the thirdFab molecule under c) are each fused at the C-terminus of the Fab heavychain to the N-terminus of one of the subunits of the Fc domain underd); andwherein the first Fab molecule under a) and the third Fab molecule underc) comprise a heavy chain variable region, particularly a humanizedheavy chain variable region, comprising the heavy chain complementaritydetermining region (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO:15 and the HCDR 3 of SEQ ID NO: 16, and a light chain variable region,particularly a humanized light chain variable region, comprising thelight chain complementarity determining region (LCDR) 1 of SEQ ID NO:17, the LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO: 19.

In another embodiment, the invention provides a T cell activatingbispecific antigen binding molecule comprising

a) a first Fab molecule which specifically binds to a first antigen;b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH or the constant domains CLand CH1 of the Fab light chain and the Fab heavy chain are replaced byeach other;c) an Fc domain composed of a first and a second subunit capable ofstable association; wherein the first antigen is CEA and the secondantigen is an activating T cell antigen, particularly CD3, moreparticularly CD3 epsilon;wherein(i) the first Fab molecule under a) is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the secondFab molecule under b), and the second Fab molecule under b) is fused atthe C-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain under c), or(ii) the second Fab molecule under b) is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the firstFab molecule under a), and the first Fab molecule under a) is fused atthe C-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain under c); andwherein the first Fab molecule under a) comprises a heavy chain variableregion, particularly a humanized heavy chain variable region, comprisingthe heavy chain complementarity determining region (HCDR) 1 of SEQ IDNO: 14, the HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO: 16, anda light chain variable region, particularly a humanized light chainvariable region, comprising the light chain complementarity determiningregion (LCDR) 1 of SEQ ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and theLCDR 3 of SEQ ID NO: 19.

In a further embodiment, the invention provides a T cell activatingbispecific antigen binding molecule comprising

a) a first Fab molecule which specifically binds to a first antigen;b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH or the constant domains CLand CH1 of the Fab light chain and the Fab heavy chain are replaced byeach other; andc) an Fc domain composed of a first and a second subunit capable ofstable association; wherein(i) the first antigen is CEA and the second antigen is an activating Tcell antigen, particularly CD3, more particularly CD3 epsilon; or(ii) the second antigen is CEA and the first antigen is an activating Tcell antigen, particularly CD3, more particularly CD3 epsilon;wherein the first Fab molecule under a) and the second Fab moleculeunder b) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c); andwherein the Fab molecule which specifically binds to CEA comprises aheavy chain variable region, particularly a humanized heavy chainvariable region, comprising the heavy chain complementarity determiningregion (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and theHCDR 3 of SEQ ID NO: 16, and a light chain variable region, particularlya humanized light chain variable region, comprising the light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 17, the LCDR 2of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO: 19.

In all of the different configurations of the T cell activatingbispecific antigen binding molecule according to the invention, theamino acid substitutions described herein, if present, may either be inthe CH1 and CL domains of the first and (if present) the third Fabmolecule, or in the CH1 and CL domains of the second Fab molecule.Preferably, they are in the CH1 and CL domains of the first and (ifpresent) the third Fab molecule. In accordance with the concept of theinvention, if amino acid substitutions as described herein are made inthe first (and, if present, the third) Fab molecule, no such amino acidsubstitutions are made in the second Fab molecule. Conversely, if aminoacid substitutions as described herein are made in the second Fabmolecule, no such amino acid substitutions are made in the first (and,if present, the third) Fab molecule. No amino acid substitutions aremade in T cell activating bispecific antigen binding moleculescomprising a Fab molecule wherein the constant domains CL and CH1 of theFab light chain and the Fab heavy chain are replaced by each other.

In particular embodiments of the T cell activating bispecific antigenbinding molecule according to the invention, particularly wherein aminoacid substitutions as described herein are made in the first (and, ifpresent, the third) Fab molecule, the constant domain CL of the first(and, if present, the third) Fab molecule is of kappa isotype. In otherembodiments of the T cell activating bispecific antigen binding moleculeaccording to the invention, particularly wherein amino acidsubstitutions as described herein are made in the second Fab molecule,the constant domain CL of the second Fab molecule is of kappa isotype.In some embodiments, the constant domain CL of the first (and, ifpresent, the third) Fab molecule and the constant domain CL of thesecond Fab molecule are of kappa isotype.

In a particular embodiment, the invention provides a T cell activatingbispecific antigen binding molecule comprising

a) a first Fab molecule which specifically binds to a first antigen;b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH of the Fab light chain andthe Fab heavy chain are replaced by each other; c) a third Fab moleculewhich specifically binds to the first antigen; andd) an Fc domain composed of a first and a second subunit capable ofstable association; wherein the first antigen is CEA and the secondantigen is an activating T cell antigen, particularly CD3, moreparticularly CD3 epsilon;wherein the third Fab molecule under c) is identical to the first Fabmolecule under a); wherein in the constant domain CL of the first Fabmolecule under a) and the third Fab molecule under c) the amino acid atposition 124 is substituted by lysine (K) (numbering according to Kabat)and the amino acid at position 123 is substituted by lysine (K) orarginine (R) (numbering according to Kabat), and wherein in the constantdomain CH1 of the first Fab molecule under a) and the third Fab moleculeunder c) the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index);wherein(i) the first Fab molecule under a) is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the secondFab molecule under b), and the second Fab molecule under b) and thethird Fab molecule under c) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder d), or(ii) the second Fab molecule under b) is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the firstFab molecule under a), and the first Fab molecule under a) and the thirdFab molecule under c) are each fused at the C-terminus of the Fab heavychain to the N-terminus of one of the subunits of the Fc domain underd); andwherein the first Fab molecule under a) and the third Fab molecule underc) comprise a heavy chain variable region, particularly a humanizedheavy chain variable region, comprising the heavy chain complementaritydetermining region (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO:15 and the HCDR 3 of SEQ ID NO: 16, and a light chain variable region,particularly a humanized light chain variable region, comprising thelight chain complementarity determining region (LCDR) 1 of SEQ ID NO:17, the LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO: 19.

In an even more particular embodiment, the invention provides a T cellactivating bispecific antigen binding molecule comprising

a) a first Fab molecule which specifically binds to a first antigen;b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH of the Fab light chain andthe Fab heavy chain are replaced by each other;c) a third Fab molecule which specifically binds to the first antigen;andd) an Fc domain composed of a first and a second subunit capable ofstable association; wherein the first antigen is CEA and the secondantigen is an activating T cell antigen, particularly CD3, moreparticularly CD3 epsilon;wherein the third Fab molecule under c) is identical to the first Fabmolecule under a); wherein in the constant domain CL of the first Fabmolecule under a) and the third Fab molecule under c) the amino acid atposition 124 is substituted by lysine (K) (numbering according to Kabat)and the amino acid at position 123 is substituted by arginine (R)(numbering according to Kabat), and wherein in the constant domain CH1of the first Fab molecule under a) and the third Fab molecule under c)the amino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex);wherein the first Fab molecule under a) is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule under b), and the second Fab molecule under b) andthe third Fab molecule under c) are each fused at the C-terminus of theFab heavy chain to the N-terminus of one of the subunits of the Fcdomain under d); and wherein the first Fab molecule under a) and thethird Fab molecule under c) comprise a heavy chain variable region,particularly a humanized heavy chain variable region, comprising theheavy chain complementarity determining region (HCDR) 1 of SEQ ID NO:14, the HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO: 16, and alight chain variable region, particularly a humanized light chainvariable region, comprising the light chain complementarity determiningregion (LCDR) 1 of SEQ ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and theLCDR 3 of SEQ ID NO: 19.

In another embodiment, the invention provides a T cell activatingbispecific antigen binding molecule comprising

a) a first Fab molecule which specifically binds to a first antigen;b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH of the Fab light chain andthe Fab heavy chain are replaced by each other; c) an Fc domain composedof a first and a second subunit capable of stable association; whereinthe first antigen is CEA and the second antigen is an activating T cellantigen, particularly CD3, more particularly CD3 epsilon;wherein in the constant domain CL of the first Fab molecule under a) theamino acid at position 124 is substituted by lysine (K) (numberingaccording to Kabat) and the amino acid at position 123 is substituted bylysine (K) or arginine (R) (numbering according to Kabat), and whereinin the constant domain CH1 of the first Fab molecule under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex);wherein(i) the first Fab molecule under a) is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the secondFab molecule under b), and the second Fab molecule under b) is fused atthe C-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain under c), or(ii) the second Fab molecule under b) is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the firstFab molecule under a), and the first Fab molecule under a) is fused atthe C-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain under c); andwherein the first Fab molecule under a) comprises a heavy chain variableregion, particularly a humanized heavy chain variable region, comprisingthe heavy chain complementarity determining region (HCDR) 1 of SEQ IDNO: 14, the HCDR 2 of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO: 16, anda light chain variable region, particularly a humanized light chainvariable region, comprising the light chain complementarity determiningregion (LCDR) 1 of SEQ ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and theLCDR 3 of SEQ ID NO: 19.

In a further embodiment, the invention provides a T cell activatingbispecific antigen binding molecule comprising

a) a first Fab molecule which specifically binds to a first antigen;b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH of the Fab light chain andthe Fab heavy chain are replaced by each other; and c) an Fc domaincomposed of a first and a second subunit capable of stable association;wherein(i) the first antigen is CEA and the second antigen is an activating Tcell antigen, particularly CD3, more particularly CD3 epsilon; or(ii) the second antigen is CEA and the first antigen is an activating Tcell antigen, particularly CD3, more particularly CD3 epsilon;wherein in the constant domain CL of the first Fab molecule under a) theamino acid at position 124 is substituted by lysine (K) (numberingaccording to Kabat) and the amino acid at position 123 is substituted bylysine (K) or arginine (R) (numbering according to Kabat), and whereinin the constant domain CH1 of the first Fab molecule under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex);wherein the first Fab molecule under a) and the second Fab moleculeunder b) are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain under c); andwherein the Fab molecule which specifically binds to CEA comprises aheavy chain variable region, particularly a humanized heavy chainvariable region, comprising the heavy chain complementarity determiningregion (HCDR) 1 of SEQ ID NO: 14, the HCDR 2 of SEQ ID NO: 15 and theHCDR 3 of SEQ ID NO: 16, and a light chain variable region, particularlya humanized light chain variable region, comprising the light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 17, the LCDR 2of SEQ ID NO: 18 and the LCDR 3 of SEQ ID NO: 19.

In particular embodiments of the T cell activating bispecific antigenbinding molecule, the Fc domain is an IgG Fc domain. In a specificembodiment, the Fc domain is an IgG₁ Fc domain. In another specificembodiment, the Fc domain is an IgG₄ Fc domain. In an even more specificembodiment, the Fc domain is an IgG₄ Fc domain comprising the amino acidsubstitution S228P (Kabat numbering). In particular embodiments the Fcdomain is a human Fc domain.

In particular embodiments, the Fc domain comprises a modificationpromoting the association of the first and the second Fc domain subunit.In a specific such embodiment, an amino acid residue in the CH3 domainof the first subunit of the Fc domain is replaced with an amino acidresidue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and an amino acid residue in the CH3 domain of the second subunit of theFc domain is replaced with an amino acid residue having a smaller sidechain volume, thereby generating a cavity within the CH3 domain of thesecond subunit within which the protuberance within the CH3 domain ofthe first subunit is positionable.

In a particular embodiment the Fc domain exhibits reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain. In certain embodiments the Fc domain isengineered to have reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a non-engineered Fc domain. Inone embodiment, the Fc domain comprises one or more amino acidsubstitution that reduces binding to an Fc receptor and/or effectorfunction.

In one embodiment, the one or more amino acid substitution in the Fedomain that reduces binding to an Fc receptor and/or effector functionis at one or more position selected from the group of L234, L235, andP329 (Kabat EU index numbering). In particular embodiments, each subunitof the Fc domain comprises three amino acid substitutions that reducebinding to an Fc receptor and/or effector function wherein said aminoacid substitutions are L234A, L235A and P329G (Kabat EU indexnumbering). In one such embodiment, the Fc domain is an IgG₁ Fc domain,particularly a human IgG₁ Fc domain. In other embodiments, each subunitof the Fc domain comprises two amino acid substitutions that reducebinding to an Fc receptor and/or effector function wherein said aminoacid substitutions are L235E and P329G (Kabat EU index numbering). Inone such embodiment, the Fc domain is an IgG₄ Fc domain, particularly ahuman IgG₄ Fc domain. In one embodiment, the Fc domain of the T cellactivating bispecific antigen binding molecule is an IgG₄ Fc domain andcomprises the amino acid substitutions L235E and S228P (SPLE) (Kabat EUindex numbering).

In one embodiment the Fc receptor is an Fcγ receptor. In one embodimentthe Fc receptor is a human Fc receptor. In one embodiment, the Fcreceptor is an activating Fc receptor. In a specific embodiment, the Fcreceptor is human FcγRIIa, FcγRI, and/or FcγRIIIa. In one embodiment,the effector function is antibody-dependent cell-mediated cytotoxicity(ADCC).

In a specific embodiment of the T cell activating bispecific antigenbinding molecule according to the invention, the antigen binding moietywhich specifically binds to an activating T cell antigen, particularlyCD3, more particularly CD3 epsilon, comprises a heavy chain variableregion comprising the heavy chain complementarity determining region(HCDR) 1 of SEQ ID NO: 4, the HCDR 2 of SEQ ID NO: 5, the HCDR 3 of SEQID NO: 6, and a light chain variable region comprising the light chaincomplementarity determining region (LCDR) 1 of SEQ ID NO: 8, the LCDR 2of SEQ ID NO: 9 and the LCDR 3 of SEQ ID NO: 10. In an even morespecific embodiment, the antigen binding moiety which specifically bindsto an activating T cell antigen, particularly CD3, more particularly CD3epsilon, comprises a heavy chain variable region comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 3 and a light chainvariable region comprising an amino acid sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 7. In some embodiments, the antigen binding moiety whichspecifically binds to an activating T cell antigen is a Fab molecule. Inone specific embodiment, the second antigen binding moiety, particularlyFab molecule, comprised in the T cell activating bispecific antigenbinding molecule according to the invention specifically binds to CD3,more particularly CD3 epsilon, and comprises the heavy chaincomplementarity determining region (CDR) 1 of SEQ ID NO: 4, the heavychain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, thelight chain CDR 1 of SEQ ID NO: 8, the light chain CDR 2 of SEQ ID NO: 9and the light chain CDR 3 of SEQ ID NO: 10. In an even more specificembodiment, said second antigen binding moiety, particularly Fabmolecule, comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 3 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 7.

In a further specific embodiment of the T cell activating bispecificantigen binding molecule according to the invention, the antigen bindingmoiety, particularly Fab molecule, which specifically binds to CEAcomprises the heavy chain complementarity determining region (CDR) 1 ofSEQ ID NO: 14, the heavy chain CDR 2 of SEQ ID NO: 15, the heavy chainCDR 3 of SEQ ID NO: 16, the light chain CDR 1 of SEQ ID NO: 17, thelight chain CDR 2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ IDNO: 19. In an even more specific embodiment, the antigen binding moiety,particularly Fab molecule, which specifically binds to CEA comprises aheavy chain variable region comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 22 and a light chain variable region comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 23. In onespecific embodiment, the first (and, if present, the third) antigenbinding moiety, particularly Fab molecule, comprised in the T cellactivating bispecific antigen binding molecule according to theinvention specifically binds to CEA, and comprises the heavy chaincomplementarity determining region (CDR) 1 of SEQ ID NO: 14, the heavychain CDR 2 of SEQ ID NO: 15, the heavy chain CDR 3 of SEQ ID NO: 16,the light chain CDR 1 of SEQ ID NO: 17, the light chain CDR 2 of SEQ IDNO: 18 and the light chain CDR 3 of SEQ ID NO: 19. In an even morespecific embodiment, said first (and, if present, said third) antigenbinding moiety, particularly Fab molecule, comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 22 anda light chain variable region comprising the amino acid sequence of SEQID NO: 23.

In a particular aspect, the invention provides a T cell activatingbispecific antigen binding molecule comprising

a) a first Fab molecule which specifically binds to a first antigen;b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH or the constant domains CLand CH1 of the Fab light chain and the Fab heavy chain are replaced byeach other;c) a third Fab molecule which specifically binds to the first antigen;andd) an Fc domain composed of a first and a second subunit capable ofstable association; wherein(i) the first antigen is CEA and the second antigen is CD3, particularlyCD3 epsilon; (ii) the first Fab molecule under a) and the third Fabmolecule under c) each comprise the heavy chain complementaritydetermining region (CDR) 1 of SEQ ID NO: 14, the heavy chain CDR 2 ofSEQ ID NO: 15, the heavy chain CDR 3 of SEQ ID NO: 16, the light chainCDR 1 of SEQ ID NO: 17, the light chain CDR 2 of SEQ ID NO: 18 and thelight chain CDR 3 of SEQ ID NO: 19, and the second Fab molecule under b)comprises the heavy chain CDR 1 of SEQ ID NO: 4, the heavy chain CDR 2of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, the light chainCDR 1 of SEQ ID NO: 8, the light chain CDR 2 of SEQ ID NO: 9 and thelight chain CDR 3 of SEQ ID NO: 10; and(iii) the first Fab molecule under a) is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the secondFab molecule under b), and the second Fab molecule under b) and thethird Fab molecule under c) are each fused at the C-terminus of the Fabheavy chain to the N-terminus of one of the subunits of the Fc domainunder d).

In one embodiment, in the second Fab molecule under b) the variabledomains VL and VH are replaced by each other and further (iv) in theconstant domain CL of the first Fab molecule under a) and the third Fabmolecule under c) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) or arginine (R), particularly byarginine (R) (numbering according to Kabat), and in the constant domainCH1 of the first Fab molecule under a) and the third Fab molecule underc) the amino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex).

According to another aspect of the invention there is provided one ormore isolated polynucleotide(s) encoding a T cell activating bispecificantigen binding molecule of the invention. The invention furtherprovides one or more expression vector(s) comprising the isolatedpolynucleotide(s) of the invention, and a host cell comprising theisolated polynucleotide(s) or the expression vector(s) of the invention.In some embodiments the host cell is a eukaryotic cell, particularly amammalian cell.

In another aspect is provided a method of producing the T cellactivating bispecific antigen binding molecule of the invention,comprising the steps of a) culturing the host cell of the inventionunder conditions suitable for the expression of the T cell activatingbispecific antigen binding molecule and b) recovering the T cellactivating bispecific antigen binding molecule. The invention alsoencompasses a T cell activating bispecific antigen binding moleculeproduced by the method of the invention.

The invention further provides a pharmaceutical composition comprisingthe T cell activating bispecific antigen binding molecule of theinvention and a pharmaceutically acceptable carrier.

Also encompassed by the invention are methods of using the T cellactivating bispecific antigen binding molecule and pharmaceuticalcomposition of the invention. In one aspect the invention provides a Tcell activating bispecific antigen binding molecule or a pharmaceuticalcomposition of the invention for use as a medicament. In one aspect isprovided a T cell activating bispecific antigen binding molecule or apharmaceutical composition according to the invention for use in thetreatment of a disease in an individual in need thereof. In a specificembodiment the disease is cancer.

Also provided is the use of a T cell activating bispecific antigenbinding molecule of the invention for the manufacture of a medicamentfor the treatment of a disease in an individual in need thereof; as wellas a method of treating a disease in an individual, comprisingadministering to said individual a therapeutically effective amount of acomposition comprising the T cell activating bispecific antigen bindingmolecule according to the invention in a pharmaceutically acceptableform. In a specific embodiment the disease is cancer. In any of theabove embodiments the individual preferably is a mammal, particularly ahuman.

The invention also provides a method for inducing lysis of a targetcell, particularly a tumor cell, comprising contacting a target cellwith a T cell activating bispecific antigen binding molecule of theinvention in the presence of a T cell, particularly a cytotoxic T cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-Z. Exemplary configurations of the T cell activating bispecificantigen binding molecules (TCBs) of the invention. (A, D) Illustrationof the “1+1 CrossMab” molecule. (B, E) Illustration of the “2+1 IgGCrossfab” molecule with alternative order of Crossfab and Fab components(“inverted”). (C, F) Illustration of the “2+1 IgG Crossfab” molecule.(G, K) Illustration of the “1+1 IgG Crossfab” molecule with alternativeorder of Crossfab and Fab components (“inverted”). (H, L) Illustrationof the “1+1 IgG Crossfab” molecule. (I, M) Illustration of the “2+1 IgGCrossfab” molecule with two CrossFabs. (J, N) Illustration of the “2+1IgG Crossfab” molecule with two CrossFabs and alternative order ofCrossfab and Fab components (“inverted”). (O, S) Illustration of the“Fab-Crossfab” molecule. (P, T) Illustration of the “Crossfab-Fab”molecule. (Q, U) Illustration of the “(Fab)₂-Crossfab” molecule. (R, V)Illustration of the “Crossfab-(Fab)₂” molecule. (W, Y) Illustration ofthe “Fab-(Crossfab)₂” molecule. (X, Z) Illustration of the“(Crossfab)₂-Fab” molecule. Black dot: optional modification in the Fcdomain promoting heterodimerization. ++, −−: amino acids of oppositecharges optionally introduced in the CH1 and CL domains. Crossfabmolecules are depicted as comprising an exchange of VH and VL regions,but may—in embodiments wherein no charge modifications are introduced inCH1 and CL domains—alternatively comprise an exchange of the CH1 and CLdomains.

FIG. 2. Binding of different humanized variants of T84.66 IgGs to cells.EC50 values, based on binding curves, were calculated by Graph Pad Prismand are presented in Table 1.

FIGS. 3A-F. Illustration of the TCBs prepared in the Examples. (A, B)Illustration of “2+1 IgG CrossFab, inverted” anti-CEA/anti-CD3 TCBmolecules with charge modifications (VH/VL exchange in CD3 binder,charge modification in CEA binder, molecule A and B). (C) Illustrationof “1+1 IgG CrossFab, inverted” anti-CEA/anti-CD3 TCB molecule withcharge modifications (VH/VL exchange in CD3 binder, charge modificationin CEA binder, molecule C). (D) Illustration of “1+1 IgG CrossMab”anti-CEA/anti-CD3 TCB molecule with charge modifications (VH/VL exchangein CD3 binder, charge modification in CEA binder, molecule D). (E)Illustration of “2+1 IgG CrossFab, inverted” anti-CEA/anti-CD3 TCBmolecule with charge modifications and longer linker (VH/VL exchange inCD3 binder, charge modification in CEA binder, molecule E). (F)Illustration of “2+1 IgG CrossFab, inverted” anti-CEA/anti-CD3 TCBmolecule without charge modifications (VH/VL exchange in CD3 binder,molecule F). EE=147E, 213E; RK=123R, 124K.

FIGS. 4A-F. CE-SDS analyses of the TCB molecules prepared in theExamples (final purified preparations). (A) Electropherogram of “2+1 IgGCrossFab, inverted” with charge modifications (VH/VL exchange in CD3binder, charge modification in CEA binder, parental murine CEA binder(T84.66); molecule A). (B) Electropherogram of “2+1 IgG CrossFab,inverted” with charge modifications (VH/VL exchange in CD3 binder,charge modification in CEA binder, humanized CEA binder; molecule B).(C) Electropherogram of “1+1 IgG CrossFab, inverted” with chargemodifications (VH/VL exchange in CD3 binder, charge modification in CEAbinder, humanized CEA binder; molecule C). (D) Electropherogram of “1+1IgG CrossMab” with charge modifications (VH/VL exchange in CD3 binder,charge modification in CEA binder, humanized CEA binder; molecule D).(E) Electropherogram of “2+1 IgG CrossFab inverted” with chargemodifications and longer linker (VH/VL exchange in CD3 binder, chargemodification in CEA binder, humanized CEA binder; molecule E). (F)Electropherogram of “2+1 IgG CrossFab, inverted” without chargemodifications (VH/VL exchange in CD3 binder, humanized CEA binder;molecule F). Lane A=non reduced, lane B=reduced.

FIGS. 5A-D. Binding of different CEA TCB formats to cells, expressingeither high levels of CEA (MKN45; A), medium levels of CEA (LS174T; B)or low levels of CEA (HT29; C), or human CD3 (Jurkat cells; D). MedianFluorescence intensities (MFI) are depicted. Error bars indicate SD oftriplicates.

FIGS. 6A-F. T cell mediated lysis of tumor cells induced by differentCEA CD3 TCB molecules, as measured by LDH release after 24 h (A-C) or 48h (D-F). Human PBMCs were used as effector cells and MKN45 (A, D),BxPC-3 (B, E) or HT29 (C, F) cells were used as target cells, at a finaleffector to target cell ratio of 10:1. Depicted are triplicates with SD.EC50 values were calculated by GraphPadPrism 5 and are given in Table 4and 5.

FIGS. 7A-H. Up-regulation of CD25 on human CD4+ (A-D) and CD8+ (E-H) Tcells after T cell-mediated lysis of CEA-expressing tumor cells inducedby different CEA CD3 TCB molecules. Human PBMCs were used as effectorcells and MKN45 (A, E), BxPC-3 (B, F) or HT29 (C, G) cells were used astarget cells, at a final effector to target cell ratio of 10:1. Resultswithout target cells are shown in (D) and (H). Percentage ofCD25-positive T cells was determined by FACS after 48 h. Depicted aretriplicates with SD.

FIGS. 8A-J. Up-regulation of CD69 on human CD4+ (A-E) and CD8+ (F-J) Tcells upon co-incubation with CEA-expressing tumor or primary epithelialcells and different CEA CD3 TCB molecules. Human PBMCs were used aseffector cells and MKN45 (A, F), LS174T (B, G), HT29 (C, H), CCD841 (D,I) cells were used as target cells, at a final effector to target cellratio of 10:1. Results without target cells are shown in (E) and (J).Percentage of CD69-positive T cells was determined by FACS after 48 h.Depicted are triplicates with SD.

FIGS. 9A-D. T cell activation and tumor cell lysis induced by differentCEA CD3 TCB molecules, as measured by FACS (percent of CD69-positiveCD8+ T cells (A, B)), or LDH (C, D) after 48 h. Human PBMCs were used aseffector cells at a final effector to target cell ratio of 10:1. Targetcells were either high CEA expressing MKN45 or low CEA-expressingprimary epithelial cells CCD841 CoN. In addition, T cell activation wasassessed in the absence of targets (“no targets”). Depicted aretriplicates with SD. A, C: CEA CD3 TCB with parental chimeric T84.66 CEAbinder (molecule A), B, D: CEA CD3 TCB with humanized CEA binder(humanized variant 1; molecule B).

FIGS. 10A-B. Jurkat-NFAT reporter cell assay to determine earlyCD3-mediated activation of Jurkats upon simultaneous binding ofdifferent CEA CD3 TCB molecules to target and Jurkat effector cells. Theintensity of CD3-mediated activation and signaling was detected bymeasuring the relative luminescence signal (RLUs). Depicted aretriplicates with SD. (A) CEA CD3 TCB with parental chimeric T84.66 CEAbinder (molecule A), (B) CEA CD3 TCB with humanized CEA binder(humanized variant 1; molecule B).

FIGS. 11A-H. T cell activation (A-D) and tumor cell lysis (E-H) inducedby different CEA CD3 TCB molecules, as measured by FACS (A-D, percent ofCD69-positive CD8+ T cells), or LDH (E-H) after 48 h. Human PBMCs wereused as effector cells at a final effector to target cell ratio of 10:1.Target cells were either medium CEA expressing BxPC-3 (A, E), lowCEA-expressing NCI-H2122 (B, F) cells or very low CEA expressingCOR-L105 (C, G) or primary epithelial cells HBEpiC (D, H). Depicted aretriplicates with SD.

FIGS. 12A-H. Proliferation of CD8+ (A-D) and CD4+ (E-H) T cells, inducedby different CEA CD3 TCB molecules, as measured by FACS after 5 days.Human PBMCs were used as effector cells at a final effector to targetcell ratio of 10:1. Target cells were either high CEA expressing MKN45(A, E), medium CEA expressing LS174T (B, F), low CEA-expressing HT29 (C,G) cells or very low CEA expressing primary epithelial cells CCD841 CoN(D, H). Depicted are triplicates with SD.

FIGS. 13A-D. Binding of different CEA CD3 TCB molecules (molecule B andmolecule E) to MKN45 (A), LS174T (B), HT29 (C) and Jurkat (D) cells, asmeasured by FACS.

FIGS. 14A-H. T cell mediated lysis of tumor cells induced by differentCEA CD3 TCB molecules (molecule B and molecule E), as measured by LDHrelease after 24 h (A-D) or 48 h (E-H). Human PBMCs were used aseffector cells and MKN45 (A, E), LS174T (B, F), HT29 (C, G), HBEpiC (D,H) cells were used as target cells, at a final effector to target cellratio of 10:1. Depicted are triplicates with SD. EC50 values werecalculated by GraphPadPrism 5 and are given in Table 6.

FIG. 15. Comparison of pharmacokinetics of CEA CD3 TCB molecule B andCEA CD3 TCB molecule X after a single i.v. bolus administration in NOGmice.

FIGS. 16A-D. Binding of different CEA CD3 TCB molecules to human CEA,expressed on MKN45 (A), LS174T (B) and HT29 (C) cells, or to human CD3,expressed on Jurkat cells (D). Depicted are triplicates and SD. EC50values, based on binding curves, were calculated by Graph Pad Prism andare presented in Table 6.

FIGS. 17A-E. Determination of antigen-dependent tumor cell lysis,induced by different CEA CD3 TCB molecules in the presence of variousCEA-positive tumor cells: (A) HCC1954, (B) NCI-H596, (C) NCI-H2122, (D)Kato III, (E) CX-1. Tumor cell lysis was determined by quantification ofLDH released from apoptotic/necrotic tumor cells. Depicted aretriplicates with SD. EC50 values of tumor cell lysis were calculated byGraph Pad Prism and are presented in Table 7.

FIGS. 18A-B. Antigen-dependent T cell activation and tumor lysis inducedby CEA CD3 TCB molecule B in the presence of CEA-positive tumor cells,but not in the presence of low CEA-expressing primary epithelia cells.Tumor cell lysis was determined by quantification of LDH released fromapoptotic/necrotic tumor cells (A). T cell activation was determined byFACS measurement of up-regulation of the early activation marker CD69 onCD4+ effector cells (B). Depicted are triplicates with SD. EC50 valuesof tumor cell lysis and T-cell activation were calculated by Graph PadPrism and are presented in Table 9 and Table 11.

FIGS. 19A-B. Antigen-dependent T cell activation and tumor lysis inducedby CEA CD3 TCB molecule B in the presence of CEA-positive tumor cells,but not in the presence of low CEA-expressing primary epithelial cells.Tumor cell lysis was determined by quantification of LDH released fromapoptotic/necrotic tumor cells (A). T cell activation was determined byFACS measurement of up-regulation of the late activation marker CD25 onCD4+ effector cells (B). Depicted are triplicates with SD. EC50 valuesof tumor cell lysis were calculated by Graph Pad Prism and are presentedin Table 10.

FIGS. 20A-B. (A) Binding of CEA CD3 TCB molecule B to transient HEK293Ttransfectants, expressing either human CEACAM5, CEACAM1 or CEACAM6.Depicted are triplicates with SD. (B) Binding of an anti-CD66 antibodyto transient HEK293T transfectants shows the transfection efficacy,respectively expression levels of the three CEACAM family members.Depicted are MFI (median fluorescence signals), based on triplicates andSD.

FIGS. 21A-C. Anti-tumor activity of CEA CD3 TCB molecule B versus CEACD3 TCB molecule X in the MKN45 model in fully humanized NOG mice.Different doses and schedules of administration were tested: (A) 2.5mg/kg twice/week, (B) 2.5 mg/kg once/week, (C) 0.5 mg/kg once/week.Black arrow indicates start of therapy. Treatment was administered for 9weeks. *p<0.05 **p<0.01; ***p<0.001 Two-tailed unpaired t-test at studytermination (day 70) (8<n<9).

FIG. 22. Simulated PK of different dose levels and schedules used in thedose-range efficacy study (Example 12). A 2-compartment model was usedand the simulation was based on the SDPK data.

FIGS. 23A-B. (A) Anti-tumor activity of CEA CD3 TCB molecule B versusCEA CD3 TCB molecule X in the MKN45 model in fully humanized NSG mice.Different doses and schedules of administration were tested. Black arrowindicates start of therapy. Treatment was administered for 4 weeks. (B)2-way ANOVA multiple comparison analysis at study termination (day 38).*p<0.05 **p<0.01; ***p<0.001; ****p<0.0001 (9<n<14).

FIG. 24. Anti-tumor activity of CEA CD3 TCB molecule B versus CEA CD3TCB molecule X (2.5 mg/kg, once a week) in the HPAF-II model in NOG micewith huPBMC transfer. Grey arrow indicates human PBMC (huPBMC) injectionand black arrow indicates start of therapy. Treatment was administeredfor 3 weeks. *p<0.05; **p<0.01; ****p<0.0001 Two-tailed unpaired t-testat study termination (day 32) (n=10).

FIG. 25. Comparison of pharmacokinetics of CEA CD3 TCB molecule B andCEA CD3 TCB molecule X after iv bolus administration in cynomolgusmonkeys. The pharmacokinetics of individual representative animals isdepicted.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are immunoglobulinsand derivatives, e.g. fragments, thereof.

The term “bispecific” means that the antigen binding molecule is able tospecifically bind to at least two distinct antigenic determinants.Typically, a bispecific antigen binding molecule comprises two antigenbinding sites, each of which is specific for a different antigenicdeterminant. In certain embodiments the bispecific antigen bindingmolecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The term “valent” as used herein denotes the presence of a specifiednumber of antigen binding sites in an antigen binding molecule. As such,the term “monovalent binding to an antigen” denotes the presence of one(and not more than one) antigen binding site specific for the antigen inthe antigen binding molecule.

An “antigen binding site” refers to the site, i.e. one or more aminoacid residues, of an antigen binding molecule which provides interactionwith the antigen. For example, the antigen binding site of an antibodycomprises amino acid residues from the complementarity determiningregions (CDRs). A native immunoglobulin molecule typically has twoantigen binding sites, a Fab molecule typically has a single antigenbinding site.

As used herein, the term “antigen binding moiety” refers to apolypeptide molecule that specifically binds to an antigenicdeterminant. In one embodiment, an antigen binding moiety is able todirect the entity to which it is attached (e.g. a second antigen bindingmoiety) to a target site, for example to a specific type of tumor cellor tumor stroma bearing the antigenic determinant. In another embodimentan antigen binding moiety is able to activate signaling through itstarget antigen, for example a T cell receptor complex antigen. Antigenbinding moieties include antibodies and fragments thereof as furtherdefined herein. Particular antigen binding moieties include an antigenbinding domain of an antibody, comprising an antibody heavy chainvariable region and an antibody light chain variable region. In certainembodiments, the antigen binding moieties may comprise antibody constantregions as further defined herein and known in the art. Useful heavychain constant regions include any of the five isotypes: α, δ, ε, γ, orμ. Useful light chain constant regions include any of the two isotypes:κ and λ.

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins referred to as antigens herein(e.g. CD3) can be any native form the proteins from any vertebratesource, including mammals such as primates (e.g. humans) and rodents(e.g. mice and rats), unless otherwise indicated. In a particularembodiment the antigen is a human protein. Where reference is made to aspecific protein herein, the term encompasses the “full-length”,unprocessed protein as well as any form of the protein that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of the protein, e.g. splice variants or allelic variants. Anexemplary human protein useful as antigen is CD3, particularly theepsilon subunit of CD3 (see UniProt no. P07766 (version 130), NCBIRefSeq no. NP_000724.1, SEQ ID NO: 1 for the human sequence; or UniProtno. Q95LI5 (version 49), NCBI GENBANK® no. BAB71849.1, SEQ ID NO: 2 forthe cynomolgus [Macaca fascicularis] sequence), or Carcinoembroynicantigen (CEA), also known as Carcinoembryonic antigen-related celladhesion molecule 5 (CEACAM5, UniProt no. P06731 (version 119), NCBIRefSeq no. NP_004354.2). In certain embodiments the T cell activatingbispecific antigen binding molecule of the invention binds to an epitopeof CD3 or CEA that is conserved among the CD3 or CEA antigens fromdifferent species.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding moiety to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance (SPR)technique (analyzed on a BIACORE® instrument) (Liljeblad et al., Glyco J17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res28, 217-229 (2002)). In one embodiment, the extent of binding of anantigen binding moiety to an unrelated protein is less than about 10% ofthe binding of the antigen binding moiety to the antigen as measured,e.g., by SPR. In certain embodiments, an antigen binding moiety thatbinds to the antigen, or an antigen binding molecule comprising thatantigen binding moiety, has a dissociation constant (K_(D)) of ≤1 μM,≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M orless, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M).

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., areceptor) and its binding partner (e.g., a ligand). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., an antigen binding moiety and an antigen, or areceptor and its ligand). The affinity of a molecule X for its partner Ycan generally be represented by the dissociation constant (K_(D)), whichis the ratio of dissociation and association rate constants (k_(off) andk_(on), respectively). Thus, equivalent affinities may comprisedifferent rate constants, as long as the ratio of the rate constantsremains the same. Affinity can be measured by well established methodsknown in the art, including those described herein. A particular methodfor measuring affinity is Surface Plasmon Resonance (SPR).

“Reduced binding”, for example reduced binding to an Fc receptor, refersto a decrease in affinity for the respective interaction, as measuredfor example by SPR. For clarity the term includes also reduction of theaffinity to zero (or below the detection limit of the analytic method),i.e. complete abolishment of the interaction. Conversely, “increasedbinding” refers to an increase in binding affinity for the respectiveinteraction.

An “activating T cell antigen” as used herein refers to an antigenicdeterminant expressed on the surface of a T lymphocyte, particularly acytotoxic T lymphocyte, which is capable of inducing T cell activationupon interaction with an antigen binding molecule. Specifically,interaction of an antigen binding molecule with an activating T cellantigen may induce T cell activation by triggering the signaling cascadeof the T cell receptor complex. In a particular embodiment theactivating T cell antigen is CD3, particularly the epsilon subunit ofCD3 (see UniProt no. P07766 (version 130), NCBI RefSeq no. NP_000724.1,SEQ ID NO: 1 for the human sequence; or UniProt no. Q95LI5 (version 49),NCBI GENBANK® no. BAB71849.1, SEQ ID NO: 2 for the cynomolgus [Macacafascicularis] sequence).

“T cell activation” as used herein refers to one or more cellularresponse of a T lymphocyte, particularly a cytotoxic T lymphocyte,selected from: proliferation, differentiation, cytokine secretion,cytotoxic effector molecule release, cytotoxic activity, and expressionof activation markers. The T cell activating bispecific antigen bindingmolecules of the invention are capable of inducing T cell activation.Suitable assays to measure T cell activation are known in the artdescribed herein.

A “target cell antigen” as used herein refers to an antigenicdeterminant presented on the surface of a target cell, for example acell in a tumor such as a cancer cell or a cell of the tumor stroma. Ina particular embodiment, the target cell antigen is CEA, particularlyhuman CEA.

As used herein, the terms “first”, “second” or “third” with respect toFab molecules etc., are used for convenience of distinguishing whenthere is more than one of each type of moiety. Use of these terms is notintended to confer a specific order or orientation of the T cellactivating bispecific antigen binding molecule unless explicitly sostated.

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

By “fused” is meant that the components (e.g. a Fab molecule and an Fcdomain subunit) are linked by peptide bonds, either directly or via oneor more peptide linkers.

As used herein, the term “single-chain” refers to a molecule comprisingamino acid monomers linearly linked by peptide bonds. In certainembodiments, one of the antigen binding moieties is a single-chain Fabmolecule, i.e. a Fab molecule wherein the Fab light chain and the Fabheavy chain are connected by a peptide linker to form a single peptidechain. In a particular such embodiment, the C-terminus of the Fab lightchain is connected to the N-terminus of the Fab heavy chain in thesingle-chain Fab molecule.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein the variable domains or the constant domains of the Fabheavy and light chain are exchanged (i.e. replaced by each other), i.e.the crossover Fab molecule comprises a peptide chain composed of thelight chain variable domain VL and the heavy chain constant domain 1 CH1(VL-CH1, in N- to C-terminal direction), and a peptide chain composed ofthe heavy chain variable domain VH and the light chain constant domainCL (VH-CL, in N- to C-terminal direction). For clarity, in a crossoverFab molecule wherein the variable domains of the Fab light chain and theFab heavy chain are exchanged, the peptide chain comprising the heavychain constant domain 1 CH1 is referred to herein as the “heavy chain”of the (crossover) Fab molecule. Conversely, in a crossover Fab moleculewherein the constant domains of the Fab light chain and the Fab heavychain are exchanged, the peptide chain comprising the heavy chainvariable domain VH is referred to herein as the “heavy chain” of the(crossover) Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fabmolecule in its natural format, i.e. comprising a heavy chain composedof the heavy chain variable and constant domains (VH-CH1, in N- toC-terminal direction), and a light chain composed of the light chainvariable and constant domains (VL-CL, in N- to C-terminal direction).

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable domain (VH), also called a variable heavy domain or a heavychain variable region, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable domain (VL), also called avariable light domain or a light chain variable region, followed by aconstant light (CL) domain, also called a light chain constant region.

The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called a (IgA), δ (IgD), ε(IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, and antibody fragments so long asthey exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and single-domain antibodies. For a review of certainantibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For areview of scFv fragments, see e.g. Pluckthun, in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab andF(ab′)₂ fragments comprising salvage receptor binding epitope residuesand having increased in vivo half-life, see U.S. Pat. No. 5,869,046.Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., ProcNatl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable domain (VL) and an antibody heavy chainvariable domain (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and by Chothia etal., J Mol Biol 196:901-917 (1987), where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table A as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe sequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular CDR given the variableregion amino acid sequence of the antibody. The CDR sequences givenherein are generally according to the Kabat definition.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table A isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table A refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein in connection with variableregion seqeunces, “Kabat numbering” refers to the numbering system setforth by Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991). Unless otherwise specified, references to the numbering ofspecific amino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

As used herein, the amino acid positions of all constant regions anddomains of the heavy and light chain are numbered according to the Kabatnumbering system described in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th ed., Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) and is referred to as“numbering according to Kabat” or “Kabat numbering” herein. Specificallythe Kabat numbering system (see pages 647-660 of Kabat, et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) is usedfor the light chain constant domain CL of kappa and lambda isotype andthe Kabat EU index numbering system (see pages 661-723) is used for theheavy chain constant domains (CH1, Hinge, CH2 and CH3), which is hereinfurther clarified by referring to “numbering according to Kabat EUindex” in this case.

The polypeptide sequences of the sequence listing are not numberedaccording to the Kabat numbering system. However, it is well within theordinary skill of one in the art to convert the numbering of thesequences of the Sequence Listing to Kabat numbering.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. Such variable domains arereferred to herein as “humanized variable region”. A humanized antibodyoptionally may comprise at least a portion of an antibody constantregion derived from a human antibody. A “humanized form” of an antibody,e.g., a non-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

The “class” of an antibody or immunoglobulin refers to the type ofconstant domain or constant region possessed by its heavy chain. Thereare five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, antibodiesproduced by host cells may undergo post-translational cleavage of one ormore, particularly one or two, amino acids from the C-terminus of theheavy chain. Therefore an antibody produced by a host cell by expressionof a specific nucleic acid molecule encoding a full-length heavy chainmay include the full-length heavy chain, or it may include a cleavedvariant of the full-length heavy chain (also referred to herein as a“cleaved variant heavy chain”). This may be the case where the final twoC-terminal amino acids of the heavy chain are glycine (G446) and lysine(K447, numbering according to Kabat EU index). Therefore, the C-terminallysine (Lys447), or the C-terminal glycine (Gly446) and lysine (K447),of the Fc region may or may not be present. Amino acid sequences ofheavy chains including Fc domains (or a subunit of an Fc domain asdefined herein) are denoted herein without C-terminal glycine-lysinedipeptide if not indicated otherwise. In one embodiment of theinvention, a heavy chain including a subunit of an Fc domain asspecified herein, comprised in a T cell activating bispecific antigenbinding molecule according to the invention, comprises an additionalC-terminal glycine-lysine dipeptide (G446 and K447, numbering accordingto EU index of Kabat). In one embodiment of the invention, a heavy chainincluding a subunit of an Fc domain as specified herein, comprised in aT cell activating bispecific antigen binding molecule according to theinvention, comprises an additional C-terminal glycine residue (G446,numbering according to EU index of Kabat). Compositions of theinvention, such as the pharmaceutical compositions described herein,comprise a population of T cell activating bispecific antigen bindingmolecules of the invention. The population of T cell activatingbispecific antigen binding molecule may comprise molecules having afull-length heavy chain and molecules having a cleaved variant heavychain. The population of T cell activating bispecific antigen bindingmolecules may consist of a mixture of molecules having a full-lengthheavy chain and molecules having a cleaved variant heavy chain, whereinat least 50%, at least 60%, at least 70%, at least 80% or at least 90%of the T cell activating bispecific antigen binding molecules have acleaved variant heavy chain. In one embodiment of the invention acomposition comprising a population of T cell activating bispecificantigen binding molecules of the invention comprises an T cellactivating bispecific antigen binding molecule comprising a heavy chainincluding a subunit of an Fc domain as specified herein with anadditional C-terminal glycine-lysine dipeptide (G446 and K447, numberingaccording to EU index of Kabat). In one embodiment of the invention acomposition comprising a population of T cell activating bispecificantigen binding molecules of the invention comprises an T cellactivating bispecific antigen binding molecule comprising a heavy chainincluding a subunit of an Fc domain as specified herein with anadditional C-terminal glycine residue (G446, numbering according to EUindex of Kabat). In one embodiment of the invention such a compositioncomprises a population of T cell activating bispecific antigen bindingmolecules comprised of molecules comprising a heavy chain including asubunit of an Fc domain as specified herein; molecules comprising aheavy chain including a subunit of a Fc domain as specified herein withan additional C-terminal glycine residue (G446, numbering according toEU index of Kabat); and molecules comprising a heavy chain including asubunit of an Fc domain as specified herein with an additionalC-terminal glycine-lysine dipeptide (G446 and K447, numbering accordingto EU index of Kabat). Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991 (seealso above). A “subunit” of an Fc domain as used herein refers to one ofthe two polypeptides forming the dimeric Fc domain, i.e. a polypeptidecomprising C-terminal constant regions of an immunoglobulin heavy chain,capable of stable self-association. For example, a subunit of an IgG Fcdomain comprises an IgG CH2 and an IgG CH3 constant domain.

A “modification promoting the association of the first and the secondsubunit of the Fc domain” is a manipulation of the peptide backbone orthe post-translational modifications of an Fc domain subunit thatreduces or prevents the association of a polypeptide comprising the Fcdomain subunit with an identical polypeptide to form a homodimer. Amodification promoting association as used herein particularly includesseparate modifications made to each of the two Fc domain subunitsdesired to associate (i.e. the first and the second subunit of the Fcdomain), wherein the modifications are complementary to each other so asto promote association of the two Fc domain subunits. For example, amodification promoting association may alter the structure or charge ofone or both of the Fc domain subunits so as to make their associationsterically or electrostatically favorable, respectively. Thus,(hetero)dimerization occurs between a polypeptide comprising the firstFc domain subunit and a polypeptide comprising the second Fc domainsubunit, which might be non-identical in the sense that furthercomponents fused to each of the subunits (e.g. antigen binding moieties)are not the same. In some embodiments the modification promotingassociation comprises an amino acid mutation in the Fc domain,specifically an amino acid substitution. In a particular embodiment, themodification promoting association comprises a separate amino acidmutation, specifically an amino acid substitution, in each of the twosubunits of the Fc domain.

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor, or increased association with another peptide. Aminoacid sequence deletions and insertions include amino- and/orcarboxy-terminal deletions and insertions of amino acids. Particularamino acid mutations are amino acid substitutions. For the purpose ofaltering e.g. the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc domain to glycinecan be indicated as 329G, G329, G₃₂₉, P329G, or Pro329Gly.

As used herein, term “polypeptide” refers to a molecule composed ofmonomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain of two ormore amino acids, and does not refer to a specific length of theproduct. Thus, peptides, dipeptides, tripeptides, oligopeptides,“protein,” “amino acid chain,” or any other term used to refer to achain of two or more amino acids, are included within the definition of“polypeptide,” and the term “polypeptide” may be used instead of, orinterchangeably with any of these terms. The term “polypeptide” is alsointended to refer to the products of post-expression modifications ofthe polypeptide, including without limitation glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide may be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Itmay be generated in any manner, including by chemical synthesis. Apolypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded.

By an “isolated” polypeptide or a variant, or derivative thereof isintended a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan be removed from its native or natural environment. Recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately receding paragraph using the ALIGN-2 computer program.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator. By a nucleic acid or polynucleotide having a nucleotidesequence at least, for example, 95% “identical” to a referencenucleotide sequence of the present invention, it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. These alterations of the reference sequence mayoccur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence. As a practicalmatter, whether any particular polynucleotide sequence is at least 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequenceof the present invention can be determined conventionally using knowncomputer programs, such as the ones discussed above for polypeptides(e.g. ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe bispecific antigen binding molecules of the present invention. Hostcells include cultured cells, e.g. mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc domain of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Humanactivating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa(CD32), and FcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies orderivatives thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “reduced ADCC” is defined as either a reduction in thenumber of target cells that are lysed in a given time, at a givenconcentration of antibody in the medium surrounding the target cells, bythe mechanism of ADCC defined above, and/or an increase in theconcentration of antibody in the medium surrounding the target cells,required to achieve the lysis of a given number of target cells in agiven time, by the mechanism of ADCC. The reduction in ADCC is relativeto the ADCC mediated by the same antibody produced by the same type ofhost cells, using the same standard production, purification,formulation and storage methods (which are known to those skilled in theart), but that has not been engineered. For example the reduction inADCC mediated by an antibody comprising in its Fc domain an amino acidsubstitution that reduces ADCC, is relative to the ADCC mediated by thesame antibody without this amino acid substitution in the Fc domain.Suitable assays to measure ADCC are well known in the art (see e.g. PCTpublication no. WO 2006/082515 or PCT publication no. WO 2012/130831).

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, T cellactivating bispecific antigen binding molecules of the invention areused to delay development of a disease or to slow the progression of adisease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The invention provides a T cell activating bispecific antigen bindingmolecule with favorable properties for therapeutic application, inparticular with improved efficacy and safety (e.g. with respect tounspecific activation of T cells or selectivity towards tumor cells overnormal cells), and improved produceability (e.g. with respect to purity,yield).

The inventors have discovered that T cell activating bispecific antigenbinding molecules comprising an antigen binding moiety with the bindingspecificity of the anti-CEA antibody T84.66 (Wagener et al., J Immunol130, 2308-(1983), Neumaier et al., J Immunol 135, 3604 (1985)) provideunexpectedly high potency in mediating killing of CEA-expressing tumorcells by T cells. Moreover, T cell activating bispecific antigen bindingmolecules comprising a novel humanized version of antibody T84.66 weresurprisingly found to exhibit improved selectivity towards tumor cellsover normal cells as compared to a T cell activating bispecific antigenbinding molecule comprising the parental T84.66 binder.

Charge Modifications

The T cell activating bispecific antigen binding molecules of theinvention may comprise amino acid substitutions in Fab moleculescomprised therein which are particularly efficient in reducingmispairing of light chains with non-matching heavy chains(Bence-Jones-type side products), which can occur in the production ofFab-based bi-/multispecific antigen binding molecules with a VH/VLexchange in one (or more, in case of molecules comprising more than twoantigen-binding Fab molecules) of their binding arms (see also PCTpublication no. WO 2015/150447, particularly the examples therein,incorporated herein by reference in its entirety).

Accordingly, in particular embodiments, the T cell activating bispecificantigen binding molecule of the invention comprises

(a) a first Fab molecule which specifically binds to a first antigen(b) a second Fab molecule which specifically binds to a second antigen,and wherein the variable domains VL and VH of the Fab light chain andthe Fab heavy chain are replaced by each other, wherein the firstantigen is an activating T cell antigen and the second antigen is CEA,or the first antigen is CEA and the second antigen is an activating Tcell antigen; and wherein

-   i) in the constant domain CL of the first Fab molecule under a) the    amino acid at position 124 is substituted by a positively charged    amino acid (numbering according to Kabat), and wherein in the    constant domain CH1 of the first Fab molecule under a) the amino    acid at position 147 or the amino acid at position 213 is    substituted by a negatively charged amino acid (numbering according    to Kabat EU index); or-   ii) in the constant domain CL of the second Fab molecule under b)    the amino acid at position 124 is substituted by a positively    charged amino acid (numbering according to Kabat), and wherein in    the constant domain CH1 of the second Fab molecule under b) the    amino acid at position 147 or the amino acid at position 213 is    substituted by a negatively charged amino acid (numbering according    to Kabat EU index).

The T cell activating bispecific antigen binding molecule does notcomprise both modifications mentioned under i) and ii). The constantdomains CL and CH1 of the second Fab molecule are not replaced by eachother (i.e. remain unexchanged).

In one embodiment of the T cell activating bispecific antigen bindingmolecule according to the invention, in the constant domain CL of thefirst Fab molecule under a) the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat) (in one preferred embodimentindependently by lysine (K) or arginine (R)), and in the constant domainCH1 of the first Fab molecule under a) the amino acid at position 147 orthe amino acid at position 213 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index).

In a further embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).

In a particular embodiment, in the constant domain CL of the first Fabmolecule under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)) and the amino acid at position 123 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and in the constant domain CH1 of the first Fabmolecule under a) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index).

In a more particular embodiment, in the constant domain CL of the firstFab molecule under a) the amino acid at position 124 is substituted bylysine (K) (numbering according to Kabat) and the amino acid at position123 is substituted by lysine (K) or arginine (R) (numbering according toKabat), and in the constant domain CH1 of the first Fab molecule undera) the amino acid at position 147 is substituted by glutamic acid (E)(numbering according to Kabat EU index) and the amino acid at position213 is substituted by glutamic acid (E) (numbering according to Kabat EUindex).

In an even more particular embodiment, in the constant domain CL of thefirst Fab molecule under a) the amino acid at position 124 issubstituted by lysine (K) (numbering according to Kabat) and the aminoacid at position 123 is substituted by arginine (R) (numbering accordingto Kabat), and in the constant domain CH1 of the first Fab moleculeunder a) the amino acid at position 147 is substituted by glutamic acid(E) (numbering according to Kabat EU index) and the amino acid atposition 213 is substituted by glutamic acid (E) (numbering according toKabat EU index).

In particular embodiments, the constant domain CL of the first Fabmolecule under a) is of kappa isotype.

Alternatively, the amino acid substitutions according to the aboveembodiments may be made in the constant domain CL and the constantdomain CH1 of the second Fab molecule under b) instead of in theconstant domain CL and the constant domain CH1 of the first Fab moleculeunder a). In particular such embodiments, the constant domain CL of thesecond Fab molecule under b) is of kappa isotype.

The T cell activating bispecific antigen binding molecule according tothe invention may further comprise a third Fab molecule whichspecifically binds to the first antigen. In particular embodiments, saidthird Fab molecule is identical to the first Fab molecule under a). Inthese embodiments, the amino acid substitutions according to the aboveembodiments will be made in the constant domain CL and the constantdomain CH1 of each of the first Fab molecule and the third Fab molecule.Alternatively, the amino acid substitutions according to the aboveembodiments may be made in the constant domain CL and the constantdomain CH1 of the second Fab molecule under b), but not in the constantdomain CL and the constant domain CH1 of the first Fab molecule and thethird Fab molecule.

In particular embodiments, the T cell activating bispecific antigenbinding molecule according to the invention further comprises an Fcdomain composed of a first and a second subunit capable of stableassociation.

T Cell Activating Bispecific Antigen Binding Molecule Formats

The components of the T cell activating bispecific antigen bindingmolecule can be fused to each other in a variety of configurations.Exemplary configurations are depicted in FIG. 1.

In particular embodiments, the antigen binding moieties comprised in theT cell activating bispecific antigen binding molecule are Fab molecules.In such embodiments, the first, second, third etc. antigen bindingmoiety may be referred to herein as first, second, third etc. Fabmolecule, respectively. Furthermore, in particular embodiments, the Tcell activating bispecific antigen binding molecule comprises an Fcdomain composed of a first and a second subunit capable of stableassociation.

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the first or the secondsubunit of the Fc domain.

In one such embodiment, the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second Fab molecule. In a specific such embodiment, the Tcell activating bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the first Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the second Fabmolecule, and the second Fab molecule is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or the second subunit ofthe Fc domain. Such a configuration is schematically depicted in FIGS.1G and 1K. Optionally, the Fab light chain of the first Fab molecule andthe Fab light chain of the second Fab molecule may additionally be fusedto each other.

In another such embodiment, the first Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain. In a specific such embodiment, the Tcell activating bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the first and the second Fab molecule are each fused at theC-terminus of the Fab heavy chain to the N-terminus of one of thesubunits of the Fc domain. Such a configuration is schematicallydepicted in FIGS. 1A and 1D. The first and the second Fab molecule maybe fused to the Fc domain directly or through a peptide linker. In aparticular embodiment the first and the second Fab molecule are eachfused to the Fc domain through an immunoglobulin hinge region. In aspecific embodiment, the immunoglobulin hinge region is a human IgG₁hinge region, particularly where the Fc domain is an IgG₁ Fc domain.

In other embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the first or second subunitof the Fc domain.

In one such embodiment, the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule. In a specific such embodiment, the Tcell activating bispecific antigen binding molecule essentially consistsof the first and the second Fab molecule, the Fc domain composed of afirst and a second subunit, and optionally one or more peptide linkers,wherein the second Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the first Fabmolecule, and the first Fab molecule is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or the second subunit ofthe Fc domain. Such a configuration is schematically depicted in FIGS.1H and 1L. Optionally, the Fab light chain of the first Fab molecule andthe Fab light chain of the second Fab molecule may additionally be fusedto each other.

The Fab molecules may be fused to the Fc domain or to each otherdirectly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) orG₄(SG₄)_(n) peptide linkers. “n” is generally an integer from 1 to 10,typically from 2 to 4. In one embodiment said peptide linker has alength of at least 5 amino acids, in one embodiment a length of 5 to100, in a further embodiment of 10 to 50 amino acids. In one embodimentsaid peptide linker is (GxS)_(n) or (GxS)_(n)G_(m) with G=glycine,S=serine, and (x=3, n=3, 4, or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3, 4or 5 and m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in afurther embodiment x=4 and n=2. In one embodiment said peptide linker is(G₄S)₂. A particularly suitable peptide linker for fusing the Fab lightchains of the first and the second Fab molecule to each other is (G₄S)₂.An exemplary peptide linker suitable for connecting the Fab heavy chainsof the first and the second Fab fragments comprises the sequence(D)-(G₄S)₂ (SEQ ID NOs 11 and 12). Another suitable such linkercomprises the sequence (G₄S)₄. Additionally, linkers may comprise (aportion of) an immunoglobulin hinge region. Particularly where a Fabmolecule is fused to the N-terminus of an Fe domain subunit, it may befused via an immunoglobulin hinge region or a portion thereof, with orwithout an additional peptide linker.

A T cell activating bispecific antigen binding molecule with a singleantigen binding moiety (such as a Fab molecule) capable of specificbinding to a target cell antigen (for example as shown in FIG. 1A, D, G,H, K, L) is useful, particularly in cases where internalization of thetarget cell antigen is to be expected following binding of a highaffinity antigen binding moiety. In such cases, the presence of morethan one antigen binding moiety specific for the target cell antigen mayenhance internalization of the target cell antigen, thereby reducing itsavailablity.

In many other cases, however, it will be advantageous to have a T cellactivating bispecific antigen binding molecule comprising two or moreantigen binding moieties (such as Fab moelcules) specific for a targetcell antigen (see examples shown in FIG. 1B, 1C, 1E, 1F, 1I, 1J, 1M or1N), for example to optimize targeting to the target site or to allowcrosslinking of target cell antigens.

Accordingly, in particular embodiments, the T cell activating bispecificantigen binding molecule of the invention further comprises a third Fabmolecule which specifically binds to the first antigen. The firstantigen preferably is the target cell antigen, i.e. CEA. In oneembodiment, the third Fab molecule is a conventional Fab molecule. Inone embodiment, the third Fab molecule is identical to the first Fabmolecule (i.e. the first and the third Fab molecule comprise the sameheavy and light chain amino acid sequences and have the same arrangementof domains (i.e. conventional or crossover)). In a particularembodiment, the second Fab molecule specifically binds to an activatingT cell antigen, particularly CD3, and the first and third Fab moleculespecifically bind to CEA.

In alternative embodiments, the T cell activating bispecific antigenbinding molecule of the invention further comprises a third Fab moleculewhich specifically binds to the second antigen. In these embodiments,the second antigen preferably is the target cell antigen, i.e. CEA. Inone such embodiment, the third Fab molecule is a crossover Fab molecule(a Fab molecule wherein the variable domains VH and VL or the constantdomains CL and CH1 of the Fab heavy and light chains areexchanged/replaced by each other). In one such embodiment, the third Fabmolecule is identical to the second Fab molecule (i.e. the second andthe third Fab molecule comprise the same heavy and light chain aminoacid sequences and have the same arrangement of domains (i.e.conventional or crossover)). In one such embodiment, the first Fabmolecule specifically binds to an activating T cell antigen,particularly CD3, and the second and third Fab molecule specificallybind to CEA.

In one embodiment, the third Fab molecule is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the first or second subunit ofthe Fc domain.

In a particular embodiment, the second and the third Fab molecule areeach fused at the C-terminus of the Fab heavy chain to the N-terminus ofone of the subunits of the Fc domain, and the first Fab molecule isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second Fab molecule. In a specific suchembodiment, the T cell activating bispecific antigen binding moleculeessentially consists of the first, the second and the third Fabmolecule, the Fc domain composed of a first and a second subunit, andoptionally one or more peptide linkers, wherein the first Fab moleculeis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the second Fab molecule, and the second Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdFab molecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain. Such a configurationis schematically depicted in FIGS. 1B and 1E (particular embodiments,wherein the third Fab molecule is a conventional Fab molecule andpreferably identical to the first Fab molecule), and FIGS. 1I and 1M(alternative embodiments, wherein the third Fab molecule is a crossoverFab molecule and preferably identical to the second Fab molecule). Thesecond and the third Fab molecule may be fused to the Fc domain directlyor through a peptide linker. In a particular embodiment the second andthe third Fab molecule are each fused to the Fc domain through animmunoglobulin hinge region. In a specific embodiment, theimmunoglobulin hinge region is a human IgG₁ hinge region, particularlywhere the Fc domain is an IgG₁ Fc domain. Optionally, the Fab lightchain of the first Fab molecule and the Fab light chain of the secondFab molecule may additionally be fused to each other.

In another embodiment, the first and the third Fab molecule are eachfused at the C-terminus of the Fab heavy chain to the N-terminus of oneof the subunits of the Fc domain, and the second Fab molecule is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the first Fab molecule. In a specific such embodiment,the T cell activating bispecific antigen binding molecule essentiallyconsists of the first, the second and the third Fab molecule, the Fcdomain composed of a first and a second subunit, and optionally one ormore peptide linkers, wherein the second Fab molecule is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first Fab molecule, and the first Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and wherein the third Fab molecule is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the secondsubunit of the Fc domain. Such a configuration is schematically depictedin FIGS. 1C and 1F (particular embodiments, wherein the third Fabmolecule is a conventional Fab molecule and preferably identical to thefirst Fab molecule) and in FIGS. 1J and 1N (alternative embodiments,wherein the third Fab molecule is a crossover Fab molecule andpreferably identical to the second Fab molecule). The first and thethird Fab molecule may be fused to the Fc domain directly or through apeptide linker. In a particular embodiment the first and the third Fabmolecule are each fused to the Fc domain through an immunoglobulin hingeregion. In a specific embodiment, the immunoglobulin hinge region is ahuman IgG₁ hinge region, particularly where the Fc domain is an IgG₁ Fcdomain. Optionally, the Fab light chain of the first Fab molecule andthe Fab light chain of the second Fab molecule may additionally be fusedto each other.

In configurations of the T cell activating bispecific antigen bindingmolecule wherein a Fab molecule is fused at the C-terminus of the Fabheavy chain to the N-terminus of each of the subunits of the Fc domainthrough an immunoglobulin hinge regions, the two Fab molecules, thehinge regions and the Fc domain essentially form an immunoglobulinmolecule. In a particular embodiment the immunoglobulin molecule is anIgG class immunoglobulin. In an even more particular embodiment theimmunoglobulin is an IgG₁ subclass immunoglobulin. In another embodimentthe immunoglobulin is an IgG₄ subclass immunoglobulin. In a furtherparticular embodiment the immunoglobulin is a human immunoglobulin. Inother embodiments the immunoglobulin is a chimeric immunoglobulin or ahumanized immunoglobulin.

In some of the T cell activating bispecific antigen binding molecule ofthe invention, the Fab light chain of the first Fab molecule and the Fablight chain of the second Fab molecule are fused to each other,optionally via a peptide linker. Depending on the configuration of thefirst and the second Fab molecule, the Fab light chain of the first Fabmolecule may be fused at its C-terminus to the N-terminus of the Fablight chain of the second Fab molecule, or the Fab light chain of thesecond Fab molecule may be fused at its C-terminus to the N-terminus ofthe Fab light chain of the first Fab molecule. Fusion of the Fab lightchains of the first and the second Fab molecule further reducesmispairing of unmatched Fab heavy and light chains, and also reduces thenumber of plasmids needed for expression of some of the T cellactivating bispecific antigen binding molecules of the invention.

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab light chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with an Fc domain subunit(VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavychain of the first Fab molecule shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In someembodiments the T cell activating bispecific antigen binding moleculefurther comprises a polypeptide wherein the Fab heavy chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the second Fab molecule(VH₍₂₎-CL₍₂₎) and the Fab light chain polypeptide of the first Fabmolecule (VL₍₁₎-CL₍₁₎). In certain embodiments the polypeptides arecovalently linked, e.g., by a disulfide bond.

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with an Fc domain subunit(VH₍₂₎-CL₍₂₎-CH2-CH3(-CH4)), and a polypeptide wherein the Fab heavychain of the first Fab molecule shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In someembodiments the T cell activating bispecific antigen binding moleculefurther comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab molecule(VL₍₂₎-CH1₍₂₎) and the Fab light chain polypeptide of the first Fabmolecule (VL₍₁₎-CL₍₁₎). In certain embodiments the polypeptides arecovalently linked, e.g., by a disulfide bond.

In some embodiments, the T cell activating bispecific antigen bindingmolecule comprises a polypeptide wherein the Fab light chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab heavy chain constant region of the second Fab molecule(i.e. the second Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain variable region is replaced by a light chainvariable region), which in turn shares a carboxy-terminal peptide bondwith the Fab heavy chain of the first Fab molecule, which in turn sharesa carboxy-terminal peptide bond with an Fc domain subunit(VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In other embodiments, the Tcell activating bispecific antigen binding molecule comprises apolypeptide wherein the Fab heavy chain of the first Fab molecule sharesa carboxy-terminal peptide bond with the Fab light chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit(VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the T cell activating bispecific antigenbinding molecule further comprises a crossover Fab light chainpolypeptide of the second Fab molecule, wherein the Fab heavy chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VH₍₂₎-CL₍₂₎), and the Fab light chain polypeptide of the firstFab molecule (VL₍₁₎-CL₍₁₎). In others of these embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain polypeptide ofthe first Fab molecule (VH₍₂₎-CL₍₂₎-VL₍₁₎-CL₍₁₎), or a polypeptidewherein the Fab light chain polypeptide of the first Fab molecule sharesa carboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VL₍₁₎-CL₍₁₎-VH₍₂₎-CL₍₂₎), as appropriate.

The T cell activating bispecific antigen binding molecule according tothese embodiments may further comprise (i) an Fc domain subunitpolypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavychain of a third Fab molecule shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab lightchain polypeptide of a third Fab molecule (VL₍₃₎-CL₍₃₎. In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In some embodiments, the T cell activating bispecific antigen bindingmolecule comprises a polypeptide wherein the Fab heavy chain variableregion of the second Fab molecule shares a carboxy-terminal peptide bondwith the Fab light chain constant region of the second Fab molecule(i.e. the second Fab molecule comprises a crossover Fab heavy chain,wherein the heavy chain constant region is replaced by a light chainconstant region), which in turn shares a carboxy-terminal peptide bondwith the Fab heavy chain of the first Fab molecule, which in turn sharesa carboxy-terminal peptide bond with an Fc domain subunit(VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎-CH2-CH3(-CH4)). In other embodiments, the Tcell activating bispecific antigen binding molecule comprises apolypeptide wherein the Fab heavy chain of the first Fab molecule sharesa carboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with an Fc domain subunit (VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎-CH2-CH3(-CH4)).

In some of these embodiments the T cell activating bispecific antigenbinding molecule further comprises a crossover Fab light chainpolypeptide of the second Fab molecule, wherein the Fab light chainvariable region of the second Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (VL₍₂₎-CH1₍₂₎), and the Fab light chain polypeptide of thefirst Fab molecule (VL₍₁₎-CL₍₁₎). In others of these embodiments the Tcell activating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule which in turn shares acarboxy-terminal peptide bond with the Fab light chain polypeptide ofthe first Fab molecule (VL₍₂₎-CH1₍₂₎-VL₍₁₎-CL₍₁₎), or a polypeptidewherein the Fab light chain polypeptide of the first Fab molecule sharesa carboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (VL₍₁₎-CL₍₁₎-VH₍₂₎-CL₍₂₎), as appropriate.

The T cell activating bispecific antigen binding molecule according tothese embodiments may further comprise (i) an Fc domain subunitpolypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide wherein the Fab heavychain of a third Fab molecule shares a carboxy-terminal peptide bondwith an Fc domain subunit (VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and the Fab lightchain polypeptide of a third Fab molecule (VL₍₃₎-CL₍₃₎. In certainembodiments the polypeptides are covalently linked, e.g., by a disulfidebond.

In some embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule. In certain such embodiments, the T cell activatingbispecific antigen binding molecule does not comprise an Fc domain. Incertain embodiments, the T cell activating bispecific antigen bindingmolecule essentially consists of the first and the second Fab molecule,and optionally one or more peptide linkers, wherein the first Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second Fab molecule. Such aconfiguration is schematically depicted in FIGS. 1O and 1S.

In other embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule. In certain such embodiments, the T cell activatingbispecific antigen binding molecule does not comprise an Fc domain. Incertain embodiments, the T cell activating bispecific antigen bindingmolecule essentially consists of the first and the second Fab molecule,and optionally one or more peptide linkers, wherein the second Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first Fab molecule. Such aconfiguration is schematically depicted in FIGS. 1P and 1T. In someembodiments, the first Fab molecule is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the secondFab molecule, and the T cell activating bispecific antigen bindingmolecule further comprises a third Fab molecule, wherein said third Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first Fab molecule. Inparticular such embodiments, said third Fab molecule is a conventionalFab molecule. In other such embodiments, said third Fab molecule is acrossover Fab molecule as described herein, i.e. a Fab molecule whereinthe variable domains VH and VL or the constant domains CL and CH1 of theFab heavy and light chains are exchanged/replaced by each other. Incertain such embodiments, the T cell activating bispecific antigenbinding molecule essentially consists of the first, the second and thethird Fab molecule, and optionally one or more peptide linkers, whereinthe first Fab molecule is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second Fab molecule, andthe third Fab molecule is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first Fab molecule. Sucha configuration is schematically depicted in FIGS. 1Q and 1U (particularembodiments, wherein the third Fab molecule is a conventional Fabmolecule and preferably identical to the first Fab molecule).

In some embodiments, the first Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond Fab molecule, and the T cell activating bispecific antigenbinding molecule further comprises a third Fab molecule, wherein saidthird Fab molecule is fused at the N-terminus of the Fab heavy chain tothe C-terminus of the Fab heavy chain of the second Fab molecule. Inparticular such embodiments, said third Fab molecule is a crossover Fabmolecule as described herein, i.e. a Fab molecule wherein the variabledomains VH and VL or the constant domains CH1 and CL of the Fab heavyand light chains are exchanged/replaced by each other. In other suchembodiments, said third Fab molecule is a conventional Fab molecule. Incertain such embodiments, the T cell activating bispecific antigenbinding molecule essentially consists of the first, the second and thethird Fab molecule, and optionally one or more peptide linkers, whereinthe first Fab molecule is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second Fab molecule, andthe third Fab molecule is fused at the N-terminus of the Fab heavy chainto the C-terminus of the Fab heavy chain of the second Fab molecule.Such a configuration is schematically depicted in FIGS. 1W and 1Y(particular embodiments, wherein the third Fab molecule is a crossoverFab molecule and preferably identical to the second Fab molecule).

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule, and the T cell activating bispecific antigen bindingmolecule further comprises a third Fab molecule, wherein said third Fabmolecule is fused at the N-terminus of the Fab heavy chain to theC-terminus of the Fab heavy chain of the first Fab molecule. Inparticular such embodiments, said third Fab molecule is a conventionalFab molecule. In other such embodiments, said third Fab molecule is acrossover Fab molecule as described herein, i.e. a Fab molecule whereinthe variable domains VH and VL or the constant domains CH1 and CL of theFab heavy and light chains are exchanged/replaced by each other. Incertain such embodiments, the T cell activating bispecific antigenbinding molecule essentially consists of the first, the second and thethird Fab molecule, and optionally one or more peptide linkers, whereinthe second Fab molecule is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the first Fabmolecule, and the third Fab molecule is fused at the N-terminus of theFab heavy chain to the C-terminus of the Fab heavy chain of the firstFab molecule. Such a configuration is schematically depicted in FIGS. 1Rand 1V (particular embodiments, wherein the third Fab molecule is aconventional Fab molecule and preferably identical to the first Fabmolecule).

In some embodiments, the second Fab molecule is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst Fab molecule, and the T cell activating bispecific antigen bindingmolecule further comprises a third Fab molecule, wherein said third Fabmolecule is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second Fab molecule. Inparticular such embodiments, said third Fab molecule is a crossover Fabmolecule as described herein, i.e. a Fab molecule wherein the variabledomains VH and VL or the constant domains CH1 and CL of the Fab heavyand light chains are exchanged/replaced by each other. In other suchembodiments, said third Fab molecule is a conventional Fab molecule. Incertain such embodiments, the T cell activating bispecific antigenbinding molecule essentially consists of the first, the second and thethird Fab molecule, and optionally one or more peptide linkers, whereinthe second Fab molecule is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the first Fabmolecule, and the third Fab molecule is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the secondFab molecule. Such a configuration is schematically depicted in FIGS. 1Xand 1Z (particular embodiments, wherein the third Fab molecule is acrossover Fab molecule and preferably identical to the first Fabmolecule).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain of the first Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain variable region of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the second Fab molecule (i.e. thesecond Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain variable region is replaced by a light chain variableregion) (VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab light chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule (VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule (VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain of the first Fab molecule, whichin turn shares a carboxy-terminal peptide bond with the Fab light chainvariable region of the second Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region)(VH₍₃₎-CH1₍₃₎-VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises the Fab light chain polypeptide of a thirdFab molecule (VL₍₃₎-CL₍₃₎.

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain of the first Fab molecule, whichin turn shares a carboxy-terminal peptide bond with the Fab heavy chainvariable region of the second Fab molecule, which in turn shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region)(VH₍₃₎-CH1₍₃₎-VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises the Fab light chain polypeptide of a thirdFab molecule (VL₍₃₎-CL₍₃₎.

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab light chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain of a third Fab molecule(VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎-VH₍₃₎-CH1₍₃₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises the Fab light chain polypeptide of a thirdFab molecule (VL₍₃₎-CL₍₃₎.

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof the second Fab molecule (i.e. the second Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain of the first Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain of a third Fab molecule(VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎-VH₍₃₎-CH1₍₃₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises the Fab light chain polypeptide of a thirdFab molecule (VL₍₃₎-CL₍₃₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain of the first Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain variable region of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab heavy chain constant region of the second Fab molecule (i.e. thesecond Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain variable region is replaced by a light chain variableregion), which in turn shares a carboxy-terminal peptide bond with theFab light chain variable region of a third Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab heavy chain constantregion of a third Fab molecule (i.e. the third Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region)(VH₍₁₎-CH1₍₁₎-VL₍₂₎-CH1₍₂₎-VL₍₃₎-CH1₍₃₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎ and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises a polypeptide wherein the Fab heavy chainvariable region of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of a third Fabmolecule (VH₍₃₎-CL₍₃₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain of the first Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain variable region of the second Fabmolecule, which in turn shares a carboxy-terminal peptide bond with theFab light chain constant region of the second Fab molecule (i.e. thesecond Fab molecule comprises a crossover Fab heavy chain, wherein theheavy chain constant region is replaced by a light chain constantregion), which in turn shares a carboxy-terminal peptide bond with theFab heavy chain variable region of a third Fab molecule, which in turnshares a carboxy-terminal peptide bond with the Fab light chain constantregion of a third Fab molecule (i.e. the third Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region)(VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎-VH₍₃₎-CL₍₃₎. In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises a polypeptide wherein the Fab light chainvariable region of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of a third Fabmolecule (VL₍₃₎-CH1₍₃₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab light chain variable region of a third Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof a third Fab molecule (i.e. the third Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain variable region isreplaced by a light chain variable region), which in turn shares acarboxy-terminal peptide bond with the Fab light chain variable regionof the second Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain variable region is replaced by a lightchain variable region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule(VL₍₃₎-CH1₍₃₎-VL₍₂₎-CH1₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab heavy chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab lightchain constant region of the second Fab molecule (VH₍₂₎-CL₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises a polypeptide wherein the Fab heavy chainvariable region of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab light chain constant region of a third Fabmolecule (VH₍₃₎-CL₍₃₎).

In certain embodiments the T cell activating bispecific antigen bindingmolecule according to the invention comprises a polypeptide wherein theFab heavy chain variable region of a third Fab molecule shares acarboxy-terminal peptide bond with the Fab light chain constant regionof a third Fab molecule (i.e. the third Fab molecule comprises acrossover Fab heavy chain, wherein the heavy chain constant region isreplaced by a light chain constant region), which in turn shares acarboxy-terminal peptide bond with the Fab heavy chain variable regionof the second Fab molecule, which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with the Fab heavy chain of the first Fab molecule(VH₍₃₎-CL₍₃₎-VH₍₂₎-CL₍₂₎-VH₍₁₎-CH1₍₁₎). In some embodiments the T cellactivating bispecific antigen binding molecule further comprises apolypeptide wherein the Fab light chain variable region of the secondFab molecule shares a carboxy-terminal peptide bond with the Fab heavychain constant region of the second Fab molecule (VL₍₂₎-CH1₍₂₎) and theFab light chain polypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎). Insome embodiments the T cell activating bispecific antigen bindingmolecule further comprises a polypeptide wherein the Fab light chainvariable region of a third Fab molecule shares a carboxy-terminalpeptide bond with the Fab heavy chain constant region of a third Fabmolecule (VL₍₃₎-CH1₍₃₎).

According to any of the above embodiments, components of the T cellactivating bispecific antigen binding molecule (e.g. Fab molecules, Fcdomain) may be fused directly or through various linkers, particularlypeptide linkers comprising one or more amino acids, typically about 2-20amino acids, that are described herein or are known in the art.Suitable, non-immunogenic peptide linkers include, for example,(G₄S)_(n), (SG₄)_(n), (G₄S)_(n) or G₄(SG₄)_(n) peptide linkers, whereinn is generally an integer from 1 to 10, typically from 2 to 4.

Fc Domain

The Fc domain of the T cell activating bispecific antigen bindingmolecule consists of a pair of polypeptide chains comprising heavy chaindomains of an immunoglobulin molecule. For example, the Fe domain of animmunoglobulin G (IgG) molecule is a dimer, each subunit of whichcomprises the CH2 and CH3 IgG heavy chain constant domains. The twosubunits of the Fc domain are capable of stable association with eachother. In one embodiment the T cell activating bispecific antigenbinding molecule of the invention comprises not more than one Fc domain.

In one embodiment according the invention the Fc domain of the T cellactivating bispecific antigen binding molecule is an IgG Fc domain. In aparticular embodiment the Fc domain is an IgG₁ Fc domain. In anotherembodiment the Fc domain is an IgG₄ Fc domain. In a more specificembodiment, the Fc domain is an IgG₄ Fc domain comprising an amino acidsubstitution at position S228 (Kabat numbering), particularly the aminoacid substitution S228P. This amino acid substitution reduces in vivoFab arm exchange of IgG₄ antibodies (see Stubenrauch et al., DrugMetabolism and Disposition 38, 84-91 (2010)). In a further particularembodiment the Fc domain is human. An exemplary sequence of a human IgG₁Fc region is given in SEQ ID NO: 13.

Fc Domain Modifications Promoting Heterodimerization

T cell activating bispecific antigen binding molecules according to theinvention comprise different Fab molecules, fused to one or the other ofthe two subunits of the Fc domain, thus the two subunits of the Fcdomain are typically comprised in two non-identical polypeptide chains.Recombinant co-expression of these polypeptides and subsequentdimerization leads to several possible combinations of the twopolypeptides. To improve the yield and purity of T cell activatingbispecific antigen binding molecules in recombinant production, it willthus be advantageous to introduce in the Fc domain of the T cellactivating bispecific antigen binding molecule a modification promotingthe association of the desired polypeptides.

Accordingly, in particular embodiments the Fc domain of the T cellactivating bispecific antigen binding molecule according to theinvention comprises a modification promoting the association of thefirst and the second subunit of the Fc domain. The site of mostextensive protein-protein interaction between the two subunits of ahuman IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in oneembodiment said modification is in the CH3 domain of the Fc domain.

There exist several approaches for modifications in the CH3 domain ofthe Fc domain in order to enforce heterodimerization, which are welldescribed e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, inall such approaches the CH3 domain of the first subunit of the Fc domainand the CH3 domain of the second subunit of the Fc domain are bothengineered in a complementary manner so that each CH3 domain (or theheavy chain comprising it) can no longer homodimerize with itself but isforced to heterodimerize with the complementarily engineered other CH3domain (so that the first and second CH3 domain heterodimerize and nohomdimers between the two first or the two second CH3 domains areformed). These different approaches for improved heavy chainheterodimerization are contemplated as different alternatives incombination with the heavy-light chain modifications (VH and VLexchange/replacement in one binding arm and the introduction ofsubstitutions of charged amino acids with opposite charges in the CH1/CLinterface) in the T cell activating bispecific antigen binding moleculeaccording to the invention which reduce light chain mispairing and BenceJones-type side products.

In a specific embodiment said modification promoting the association ofthe first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). Accordingly, in a particularembodiment, in the CH3 domain of the first subunit of the Fc domain ofthe T cell activating bispecific antigen binding molecule an amino acidresidue is replaced with an amino acid residue having a larger sidechain volume, thereby generating a protuberance within the CH3 domain ofthe first subunit which is positionable in a cavity within the CH3domain of the second subunit, and in the CH3 domain of the secondsubunit of the Fc domain an amino acid residue is replaced with an aminoacid residue having a smaller side chain volume, thereby generating acavity within the CH3 domain of the second subunit within which theprotuberance within the CH3 domain of the first subunit is positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), and tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V).

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of theFc domain (the “knobs” subunit) the threonine residue at position 366 isreplaced with a tryptophan residue (T366W), and in the CH3 domain of thesecond subunit of the Fc domain (the “hole” subunit) the tyrosineresidue at position 407 is replaced with a valine residue (Y407V). Inone embodiment, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A) (numberings according to Kabat EU index).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C) or the glutamic acid residue at position 356 isreplaced with a cysteine residue (E356C), and in the second subunit ofthe Fc domain additionally the tyrosine residue at position 349 isreplaced by a cysteine residue (Y349C) (numberings according to Kabat EUindex). Introduction of these two cysteine residues results in formationof a disulfide bridge between the two subunits of the Fc domain, furtherstabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In a particular embodiment, the first subunit of the Fc domain comprisesamino acid substitutions S354C and T366W, and the second subunit of theFc domain comprises amino acid substitutions Y349C, T366S, L368A andY407V (numbering according to Kabat EU index).

In a particular embodiment the Fab molecule which specifically binds anactivating T cell antigen is fused (optionally via a Fab molecule whichspecifically binds to a target cell antigen) to the first subunit of theFc domain (comprising the “knob” modification). Without wishing to bebound by theory, fusion of the Fab molecule which specifically binds anactivating T cell antigen to the knob-containing subunit of the Fcdomain will (further) minimize the generation of antigen bindingmolecules comprising two Fab molecules which bind to an activating Tcell antigen (steric clash of two knob-containing polypeptides).

Other techniques of CH3-modification for enforcing theheterodimerization are contemplated as alternatives according to theinvention and are described e.g. in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304,WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO2013/096291.

In one embodiment the heterodimerization approach described in EP1870459 A1, is used alternatively. This approach is based on theintroduction of charged amino acids with opposite charges at specificamino acid positions in the CH3/CH3 domain interface between the twosubunits of the Fc domain. One preferred embodiment for the T cellactivating bispecific antigen binding molecule of the invention areamino acid mutations R409D; K370E in one of the two CH3 domains (of theFc domain) and amino acid mutations D399K; E357K in the other one of theCH3 domains of the Fc domain (numbering according to Kabat EU index).

In another embodiment the T cell activating bispecific antigen bindingmolecule of the invention comprises amino acid mutation T366W in the CH3domain of the first subunit of the Fc domain and amino acid mutationsT366S, L368A, Y407V in the CH3 domain of the second subunit of the Fcdomain, and additionally amino acid mutations R409D; K370E in the CH3domain of the first subunit of the Fc domain and amino acid mutationsD399K; E357K in the CH3 domain of the second subunit of the Fc domain(numberings according to Kabat EU index).

In another embodiment T cell activating bispecific antigen bindingmolecule of the invention comprises amino acid mutations S354C, T366W inthe CH3 domain of the first subunit of the Fc domain and amino acidmutations Y349C, T366S, L368A, Y407V in the CH3 domain of the secondsubunit of the Fc domain, or said T cell activating bispecific antigenbinding molecule comprises amino acid mutations Y349C, T366W in the CH3domain of the first subunit of the Fc domain and amino acid mutationsS354C, T366S, L368A, Y407V in the CH3 domains of the second subunit ofthe Fc domain and additionally amino acid mutations R409D; K370E in theCH3 domain of the first subunit of the Fc domain and amino acidmutations D399K; E357K in the CH3 domain of the second subunit of the Fcdomain (all numberings according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2013/157953 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366K and a second CH3 domain comprisesamino acid mutation L351D (numberings according to Kabat EU index). In afurther embodiment the first CH3 domain comprises further amino acidmutation L351K. In a further embodiment the second CH3 domain comprisesfurther an amino acid mutation selected from Y349E, Y349D and L368E(preferably L368E) (numberings according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2012/058768 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutations L351Y, Y407A and a second CH3 domaincomprises amino acid mutations T366A, K409F. In a further embodiment thesecond CH3 domain comprises a further amino acid mutation at positionT411, D399, S400, F405, N390, or K392, e.g. selected from a) T411N,T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y orD399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S,F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L,K392F or K392E (numberings according to Kabat EU index). In a furtherembodiment a first CH3 domain comprises amino acid mutations L351Y,Y407A and a second CH3 domain comprises amino acid mutations T366V,K409F. In a further embodiment a first CH3 domain comprises amino acidmutation Y407A and a second CH3 domain comprises amino acid mutationsT366A, K409F. In a further embodiment the second CH3 domain furthercomprises amino acid mutations K392E, T411E, D399R and S400R (numberingsaccording to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2011/143545 is used alternatively, e.g. with the amino acid modificationat a position selected from the group consisting of 368 and 409(numbering according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO2011/090762, which also uses the knobs-into-holes technology describedabove, is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366W and a second CH3 domain comprisesamino acid mutation Y407A. In one embodiment a first CH3 domaincomprises amino acid mutation T366Y and a second CH3 domain comprisesamino acid mutation Y407T (numberings according to Kabat EU index).

In one embodiment the T cell activating bispecific antigen bindingmolecule or its Fc domain is of IgG₂ subclass and the heterodimerizationapproach described in WO 2010/129304 is used alternatively.

In an alternative embodiment a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication no. WO 2009/089004. Generally, this method involvesreplacement of one or more amino acid residues at the interface of thetwo Fc domain subunits by charged amino acid residues so that homodimerformation becomes electrostatically unfavorable but heterodimerizationelectrostatically favorable. In one such embodiment a first CH3 domaincomprises amino acid substitution of K392 or N392 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D),preferably K392D or N392D) and a second CH3 domain comprises amino acidsubstitution of D399, E356, D356, or E357 with a positively chargedamino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K, and more preferably D399K and E356K). In a furtherembodiment the first CH3 domain further comprises amino acidsubstitution of K409 or R409 with a negatively charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). Ina further embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (allnumberings according to Kabat EU index).

In yet a further embodiment the heterodimerization approach described inWO 2007/147901 is used alternatively. In one embodiment a first CH3domain comprises amino acid mutations K253E, D282K, and K322D and asecond CH3 domain comprises amino acid mutations D239K, E240K, and K292D(numberings according to Kabat EU index).

In still another embodiment the heterodimerization approach described inWO 2007/110205 can be used alternatively.

In one embodiment, the first subunit of the Fc domain comprises aminoacid substitutions K392D and K409D, and the second subunit of the Fcdomain comprises amino acid substitutions D356K and D399K (numberingaccording to Kabat EU index).

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to the T cell activating bispecific antigenbinding molecule favorable pharmacokinetic properties, including a longserum half-life which contributes to good accumulation in the targettissue and a favorable tissue-blood distribution ratio. At the same timeit may, however, lead to undesirable targeting of the T cell activatingbispecific antigen binding molecule to cells expressing Fc receptorsrather than to the preferred antigen-bearing cells. Moreover, theco-activation of Fc receptor signaling pathways may lead to cytokinerelease which, in combination with the T cell activating properties andthe long half-life of the antigen binding molecule, results in excessiveactivation of cytokine receptors and severe side effects upon systemicadministration. Activation of (Fc receptor-bearing) immune cells otherthan T cells may even reduce efficacy of the T cell activatingbispecific antigen binding molecule due to the potential destruction ofT cells e.g. by NK cells.

Accordingly, in particular embodiments, the Fc domain of the T cellactivating bispecific antigen binding molecules according to theinvention exhibits reduced binding affinity to an Fc receptor and/orreduced effector function, as compared to a native IgG₁ Fc domain. Inone such embodiment the Fc domain (or the T cell activating bispecificantigen binding molecule comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG₁ Fc domain (or a T cell activating bispecificantigen binding molecule comprising a native IgG₁ Fc domain), and/orless than 50%, preferably less than 20%, more preferably less than 10%and most preferably less than 5% of the effector function, as comparedto a native IgG₁ Fc domain domain (or a T cell activating bispecificantigen binding molecule comprising a native IgG₁ Fc domain). In oneembodiment, the Fc domain domain (or the T cell activating bispecificantigen binding molecule comprising said Fc domain) does notsubstantially bind to an Fc receptor and/or induce effector function. Ina particular embodiment the Fc receptor is an Fcγ receptor. In oneembodiment the Fc receptor is a human Fc receptor. In one embodiment theFc receptor is an activating Fc receptor. In a specific embodiment theFc receptor is an activating human Fcγ receptor, more specifically humanFcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In oneembodiment the effector function is one or more selected from the groupof CDC, ADCC, ADCP, and cytokine secretion. In a particular embodimentthe effector function is ADCC. In one embodiment the Fc domain domainexhibits substantially similar binding affinity to neonatal Fc receptor(FcRn), as compared to a native IgG₁ Fc domain domain. Substantiallysimilar binding to FcRn is achieved when the Fc domain (or the T cellactivating bispecific antigen binding molecule comprising said Fcdomain) exhibits greater than about 70%, particularly greater than about80%, more particularly greater than about 90% of the binding affinity ofa native IgG₁ Fc domain (or the T cell activating bispecific antigenbinding molecule comprising a native IgG₁ Fc domain) to FcRn.

In certain embodiments the Fe domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain of the T cell activating bispecific antigen binding moleculecomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function.Typically, the same one or more amino acid mutation is present in eachof the two subunits of the Fc domain. In one embodiment the amino acidmutation reduces the binding affinity of the Fc domain to an Fcreceptor. In one embodiment the amino acid mutation reduces the bindingaffinity of the Fc domain to an Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In embodiments where there is more than oneamino acid mutation that reduces the binding affinity of the Fc domainto the Fc receptor, the combination of these amino acid mutations mayreduce the binding affinity of the Fc domain to an Fc receptor by atleast 10-fold, at least 20-fold, or even at least 50-fold. In oneembodiment the T cell activating bispecific antigen binding moleculecomprising an engineered Fc domain exhibits less than 20%, particularlyless than 10%, more particularly less than 5% of the binding affinity toan Fc receptor as compared to a T cell activating bispecific antigenbinding molecule comprising a non-engineered Fc domain. In a particularembodiment the Fc receptor is an Fcγ receptor. In some embodiments theFc receptor is a human Fc receptor. In some embodiments the Fc receptoris an activating Fc receptor. In a specific embodiment the Fc receptoris an activating human Fcγ receptor, more specifically human FcγRIIIa,FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, bindingto each of these receptors is reduced. In some embodiments bindingaffinity to a complement component, specifically binding affinity toC1q, is also reduced. In one embodiment binding affinity to neonatal Fcreceptor (FcRn) is not reduced. Substantially similar binding to FcRn,i.e. preservation of the binding affinity of the Fc domain to saidreceptor, is achieved when the Fc domain (or the T cell activatingbispecific antigen binding molecule comprising said Fc domain) exhibitsgreater than about 70% of the binding affinity of a non-engineered formof the Fc domain (or the T cell activating bispecific antigen bindingmolecule comprising said non-engineered form of the Fc domain) to FcRn.The Fc domain, or T cell activating bispecific antigen binding moleculesof the invention comprising said Fc domain, may exhibit greater thanabout 80% and even greater than about 90% of such affinity. In certainembodiments the Fc domain of the T cell activating bispecific antigenbinding molecule is engineered to have reduced effector function, ascompared to a non-engineered Fc domain. The reduced effector functioncan include, but is not limited to, one or more of the following:reduced complement dependent cytotoxicity (CDC), reducedantibody-dependent cell-mediated cytotoxicity (ADCC), reducedantibody-dependent cellular phagocytosis (ADCP), reduced cytokinesecretion, reduced immune complex-mediated antigen uptake byantigen-presenting cells, reduced binding to NK cells, reduced bindingto macrophages, reduced binding to monocytes, reduced binding topolymorphonuclear cells, reduced direct signaling inducing apoptosis,reduced crosslinking of target-bound antibodies, reduced dendritic cellmaturation, or reduced T cell priming. In one embodiment the reducedeffector function is one or more selected from the group of reduced CDC,reduced ADCC, reduced ADCP, and reduced cytokine secretion. In aparticular embodiment the reduced effector function is reduced ADCC. Inone embodiment the reduced ADCC is less than 20% of the ADCC induced bya non-engineered Fc domain (or a T cell activating bispecific antigenbinding molecule comprising a non-engineered Fc domain).

In one embodiment the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment the Fc domain comprises anamino acid substitution at a position selected from the group of E233,L234, L235, N297, P331 and P329 (numberings according to Kabat EUindex). In a more specific embodiment the Fc domain comprises an aminoacid substitution at a position selected from the group of L234, L235and P329 (numberings according to Kabat EU index). In some embodimentsthe Fc domain comprises the amino acid substitutions L234A and L235A(numberings according to Kabat EU index). In one such embodiment, the Fcdomain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain. In oneembodiment the Fc domain comprises an amino acid substitution atposition P329. In a more specific embodiment the amino acid substitutionis P329A or P329G, particularly P329G (numberings according to Kabat EUindex). In one embodiment the Fc domain comprises an amino acidsubstitution at position P329 and a further amino acid substitution at aposition selected from E233, L234, L235, N297 and P331 (numberingsaccording to Kabat EU index). In a more specific embodiment the furtheramino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D orP331S. In particular embodiments the Fc domain comprises amino acidsubstitutions at positions P329, L234 and L235 (numberings according toKabat EU index). In more particular embodiments the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”). In onesuch embodiment, the Fc domain is an IgG₁ Fc domain, particularly ahuman IgG₁ Fc domain. The “P329G LALA” combination of amino acidsubstitutions almost completely abolishes Fcγ receptor (as well ascomplement) binding of a human IgG₁ Fc domain, as described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety. WO 2012/130831 also describes methods of preparing such mutantFc domains and methods for determining its properties such as Fcreceptor binding or effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments the Fc domain of the T cell activating bispecificantigen binding molecules of the invention is an IgG₄ Fc domain,particularly a human IgG₄ Fc domain. In one embodiment the IgG₄ Fcdomain comprises amino acid substitutions at position S228, specificallythe amino acid substitution S228P (numberings according to Kabat EUindex). To further reduce its binding affinity to an Fc receptor and/orits effector function, in one embodiment the IgG₄ Fc domain comprises anamino acid substitution at position L235, specifically the amino acidsubstitution L235E (numberings according to Kabat EU index). In anotherembodiment, the IgG₄ Fc domain comprises an amino acid substitution atposition P329, specifically the amino acid substitution P329G(numberings according to Kabat EU index). In a particular embodiment,the IgG₄ Fc domain comprises amino acid substitutions at positions S228,L235 and P329, specifically amino acid substitutions S228P, L235E andP329G (numberings according to Kabat EU index). Such IgG₄ Fc domainmutants and their Fc receptor binding properties are described in PCTpublication no. WO 2012/130831, incorporated herein by reference in itsentirety. In a particular embodiment the Fc domain exhibiting reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a native IgG₁ Fc domain, is a human IgG₁ Fc domaincomprising the amino acid substitutions L234A, L235A and optionallyP329G, or a human IgG₄ Fc domain comprising the amino acid substitutionsS228P, L235E and optionally P329G (numberings according to Kabat EUindex).

In certain embodiments N-glycosylation of the Fc domain has beeneliminated. In one such embodiment the Fc domain comprises an amino acidmutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D)(numberings according to Kabat EU index).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056) (numberings according to Kabat EU index). SuchFc mutants include Fc mutants with substitutions at two or more of aminoacid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine(U.S. Pat. No. 7,332,581).

Mutant Fe domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIACORE® instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor.

Effector function of an Fc domain, or a T cell activating bispecificantigen binding molecule comprising an Fc domain, can be measured bymethods known in the art. A suitable assay for measuring ADCC isdescribed herein. Other examples of in vitro assays to assess ADCCactivity of a molecule of interest are described in U.S. Pat. No.5,500,362; Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986)and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g. in a animal model such as that disclosed in Clynes et al.,Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the T cell activating bispecificantigen binding molecule is able to bind C1q and hence has CDC activity.See e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro et al., J Immunol Methods202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Craggand Glennie, Blood 103, 2738-2743 (2004)).

Antigen Binding Moieties

The antigen binding molecule of the invention is bispecific, i.e. itcomprises at least two antigen binding moieties capable of specificbinding to two distinct antigenic determinants. According to particularembodiments of the invention, the antigen binding moieties are Fabmolecules (i.e. antigen binding domains composed of a heavy and a lightchain, each comprising a variable and a constant domain). In oneembodiment said Fab molecules are human. In another embodiment said Fabmolecules are humanized. In yet another embodiment said Fab moleculescomprise human heavy and light chain constant domains.

Preferably, at least one of the antigen binding moieties is a crossoverFab molecule. Such modification reduces mispairing of heavy and lightchains from different Fab molecules, thereby improving the yield andpurity of the T cell activating bispecific antigen binding molecule ofthe invention in recombinant production. In a particular crossover Fabmolecule useful for the T cell activating bispecific antigen bindingmolecule of the invention, the variable domains of the Fab light chainand the Fab heavy chain (VL and VH, respectively) are exchanged. Evenwith this domain exchange, however, the preparation of the T cellactivating bispecific antigen binding molecule may comprise certain sideproducts due to a so-called Bence Jones-type interaction betweenmispaired heavy and light chains (see Schaefer et al, PNAS, 108 (2011)11187-11191). To further reduce mispairing of heavy and light chainsfrom different Fab molecules and thus increase the purity and yield ofthe desired T cell activating bispecific antigen binding molecule,according to the present invention charged amino acids with oppositecharges may be introduced at specific amino acid positions in the CH1and CL domains of either the Fab molecule(s) specifically binding to atarget cell antigen, or the Fab molecule specifically binding to anactivating T cell antigen. Charge modifications are made either in theconventional Fab molecule(s) comprised in the T cell activatingbispecific antigen binding molecule (such as shown e.g. in FIGS. 1 A-C,G-J), or in the VH/VL crossover Fab molecule(s) comprised in the T cellactivating bispecific antigen binding molecule (such as shown e.g. inFIG. 1 D-F, K-N) (but not in both). In particular embodiments, thecharge modifications are made in the conventional Fab molecule(s)comprised in the T cell activating bispecific antigen binding molecule(which in particular embodiments specifically bind(s) to the target cellantigen).

In a particular embodiment according to the invention, the T cellactivating bispecific antigen binding molecule is capable ofsimultaneous binding to a target cell antigen, particularly a tumor cellantigen, and an activating T cell antigen, particularly CD3. In oneembodiment, the T cell activating bispecific antigen binding molecule iscapable of crosslinking a T cell and a target cell by simultaneousbinding to a target cell antigen and an activating T cell antigen. In aneven more particular embodiment, such simultaneous binding results inlysis of the target cell, particularly a tumor cell. In one embodiment,such simultaneous binding results in activation of the T cell. In otherembodiments, such simultaneous binding results in a cellular response ofa T lymphocyte, particularly a cytotoxic T lymphocyte, selected from thegroup of: proliferation, differentiation, cytokine secretion, cytotoxiceffector molecule release, cytotoxic activity, and expression ofactivation markers. In one embodiment, binding of the T cell activatingbispecific antigen binding molecule to the activating T cell antigen,particularly CD3, without simultaneous binding to the target cellantigen does not result in T cell activation.

In one embodiment, the T cell activating bispecific antigen bindingmolecule is capable of re-directing cytotoxic activity of a T cell to atarget cell. In a particular embodiment, said re-direction isindependent of MHC-mediated peptide antigen presentation by the targetcell and and/or specificity of the T cell.

Particularly, a T cell according to any of the embodiments of theinvention is a cytotoxic T cell. In some embodiments the T cell is aCD4⁺ or a CD8⁺ T cell, particularly a CD8⁺ T cell.

Activating T Cell Antigen Binding Moiety

The T cell activating bispecific antigen binding molecule of theinvention comprises at least one antigen binding moiety, particularly aFab molecule, which specifically binds to an activating T cell antigen(also referred to herein as an “activating T cell antigen bindingmoiety, or activating T cell antigen binding Fab molecule”). In aparticular embodiment, the T cell activating bispecific antigen bindingmolecule comprises not more than one antigen binding moiety capable ofspecific binding to an activating T cell antigen. In one embodiment theT cell activating bispecific antigen binding molecule providesmonovalent binding to the activating T cell antigen.

In particular embodiments, the antigen binding moiety which specificallybinds an activating T cell antigen is a crossover Fab molecule asdescribed herein, i.e. a Fab molecule wherein the variable domains VHand VL or the constant domains CH1 and CL of the Fab heavy and lightchains are exchanged/replaced by each other. In such embodiments, theantigen binding moiety(ies) which specifically binds a target cellantigen is preferably a conventional Fab molecule. In embodiments wherethere is more than one antigen binding moiety, particularly Fabmolecule, which specifically binds to a target cell antigen comprised inthe T cell activating bispecific antigen binding molecule, the antigenbinding moiety which specifically binds to an activating T cell antigenpreferably is a crossover Fab molecule and the antigen binding moietieswhich specifically bind to a target cell antigen are conventional Fabmolecules.

In alternative embodiments, the antigen binding moiety whichspecifically binds an activating T cell antigen is a conventional Fabmolecule. In such embodiments, the antigen binding moiety(ies) whichspecifically binds a target cell antigen is a crossover Fab molecule asdescribed herein, i.e. a Fab molecule wherein the variable domains VHand VL or the constant domains CH1 and CL of the Fab heavy and lightchains are exchanged/replaced by each other.

In a particular embodiment the activating T cell antigen is CD3,particularly human CD3 (SEQ ID NO: 1) or cynomolgus CD3 (SEQ ID NO: 2),most particularly human CD3. In a particular embodiment the activating Tcell antigen binding moiety is cross-reactive for (i.e. specificallybinds to) human and cynomolgus CD3. In some embodiments, the activatingT cell antigen is the epsilon subunit of CD3 (CD3 epsilon).

In some embodiments, the activating T cell antigen binding moietyspecifically binds to CD3, particularly CD3 epsilon, and comprises atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6and at least one light chain CDR selected from the group of SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region comprising theheavy chain CDR1 of SEQ ID NO: 4, the heavy chain CDR2 of SEQ ID NO: 5,the heavy chain CDR3 of SEQ ID NO: 6, and a light chain variable regioncomprising the light chain CDR1 of SEQ ID NO: 8, the light chain CDR2 ofSEQ ID NO: 9, and the light chain CDR3 of SEQ ID NO: 10.

In another embodiment the CD3 binding antigen binding moiety,particularly Fab molecule, comprises a heavy chain variable regioncomprising the heavy chain CDR1 of SEQ ID NO: 4, the heavy chain CDR2 ofSEQ ID NO: 46, the heavy chain CDR3 of SEQ ID NO: 6, and a light chainvariable region comprising the light chain CDR1 of SEQ ID NO: 47, thelight chain CDR2 of SEQ ID NO: 9, and the light chain CDR3 of SEQ ID NO:10.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3and a light chain variable region sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 3 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 7.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises the heavy chain variable region sequence of SEQID NO: 3 and the light chain variable region sequence of SEQ ID NO: 7.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:48 and a light chain variable region sequence that is at least about95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 48 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 49.

In one embodiment the CD3 binding antigen binding moiety, particularlyFab molecule, comprises the heavy chain variable region sequence of SEQID NO: 48 and the light chain variable region sequence of SEQ ID NO: 49.

Target Cell Antigen Binding Moiety

The T cell activating bispecific antigen binding molecule of theinvention comprises at least one antigen binding moiety, particularly aFab molecule, which specifically binds to CEA (target cell antigen). Incertain embodiments, the T cell activating bispecific antigen bindingmolecule comprises two antigen binding moieties, particularly Fabmolecules, which specifically bind to CEA.

In a particular such embodiment, each of these antigen binding moietiesspecifically binds to the same antigenic determinant. In an even moreparticular embodiment, all of these antigen binding moieties areidentical, i.e. they comprise the same amino acid sequences includingthe same amino acid substitutions in the CH1 and CL domain as describedherein (if any). In one embodiment, the T cell activating bispecificantigen binding molecule comprises an immunoglobulin molecule whichspecifically binds to CEA. In one embodiment the T cell activatingbispecific antigen binding molecule comprises not more than two antigenbinding moieties, particularly Fab molecules, which specifically bind toCEA.

In particular embodiments, the antigen binding moiety(ies) whichspecifically bind to CEA is/are a conventional Fab molecule. In suchembodiments, the antigen binding moiety(ies) which specifically binds anactivating T cell antigen is a crossover Fab molecule as describedherein, i.e. a Fab molecule wherein the variable domains VH and VL orthe constant domains CH1 and CL of the Fab heavy and light chains areexchanged/replaced by each other.

In alternative embodiments, the antigen binding moiety(ies) whichspecifically bind to CEA is/are a crossover Fab molecule as describedherein, i.e. a Fab molecule wherein the variable domains VH and VL orthe constant domains CH1 and CL of the Fab heavy and light chains areexchanged/replaced by each other. In such embodiments, the antigenbinding moiety(ies) which specifically binds an activating T cellantigen is a conventional Fab molecule.

The CEA binding moiety is able to direct the T cell activatingbispecific antigen binding molecule to a target site, for example to aspecific type of tumor cell that expresses CEA.

In one embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CEA comprises a heavy chainvariable region comprising the heavy chain complementarity determiningregion (CDR) 1 of SEQ ID NO: 14, the heavy chain CDR 2 of SEQ ID NO: 15,and the heavy chain CDR 3 of SEQ ID NO: 16, and a light chain variableregion comprising the light chain CDR 1 of SEQ ID NO: 17, the lightchain CDR 2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ ID NO: 19.In a further embodiment, the antigen binding moiety, particularly Fabmolecule, which specifically binds to CEA comprises a heavy chainvariable region that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 22, and a light chain variable region that isat least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ IDNO: 23. In still a further embodiment, the antigen binding moiety,particularly Fab molecule, which specifically binds to CEA comprises theheavy chain variable region sequence of SEQ ID NO: 22, and the lightchain variable region sequence of SEQ ID NO: 23. In a particularembodiment, the T cell activating bispecific antigen binding moleculecomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 34, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:36, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 37, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 38.In a further particular embodiment, the T cell activating bispecificantigen binding molecule comprises a polypeptide sequence of SEQ ID NO:34, a polypeptide sequence of SEQ ID NO: 36, a polypeptide sequence ofSEQ ID NO: 37 and a polypeptide sequence of SEQ ID NO: 38. In anotherembodiment, the T cell activating bispecific antigen binding moleculecomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 34, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:37, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 38, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 39.In a further embodiment, the the T cell activating bispecific antigenbinding molecule comprises a polypeptide sequence of SEQ ID NO: 34, apolypeptide sequence of SEQ ID NO: 37, a polypeptide sequence of SEQ IDNO: 38 and a polypeptide sequence of SEQ ID NO: 39. In still anotherembodiment, the T cell activating bispecific antigen binding moleculecomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 34, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:36, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 38, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 40.In a further embodiment, the the T cell activating bispecific antigenbinding molecule comprises a polypeptide sequence of SEQ ID NO: 34, apolypeptide sequence of SEQ ID NO: 36, a polypeptide sequence of SEQ IDNO: 38 and a polypeptide sequence of SEQ ID NO: 40. In a furtherembodiment, the T cell activating bispecific antigen binding moleculecomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 34, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:36, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 38, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 41.In a further embodiment, the the T cell activating bispecific antigenbinding molecule comprises a polypeptide sequence of SEQ ID NO: 34, apolypeptide sequence of SEQ ID NO: 36, a polypeptide sequence of SEQ IDNO: 38 and a polypeptide sequence of SEQ ID NO: 41. In yet anotherembodiment, the T cell activating bispecific antigen binding moleculecomprises a polypeptide that is at least 95%, 96%, 97%, 98%, or 99%identical to the sequence of SEQ ID NO: 34, a polypeptide that is atleast 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:50, a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identicalto the sequence of SEQ ID NO: 51, and a polypeptide that is at least95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 52.In a further embodiment, the the T cell activating bispecific antigenbinding molecule comprises a polypeptide sequence of SEQ ID NO: 34, apolypeptide sequence of SEQ ID NO: 50, a polypeptide sequence of SEQ IDNO: 51 and a polypeptide sequence of SEQ ID NO: 52.

CEA Antibodies

In one aspect the invention provides an antibody, particularly ahumanized antibody, which specifically binds to CEA, wherein saidantibody comprises a heavy chain variable region comprising the heavychain complementarity determining region (CDR) 1 of SEQ ID NO: 14, theheavy chain CDR 2 of SEQ ID NO: 15, and the heavy chain CDR 3 of SEQ IDNO: 16, and a light chain variable region comprising the light chain CDR1 of SEQ ID NO: 17, the light chain CDR 2 of SEQ ID NO: 18 and the lightchain CDR 3 of SEQ ID NO: 19. In one embodiment, the antibody comprisesa heavy chain variable region that is at least 95%, 96%, 97%, 98%, or99% identical to the sequence of SEQ ID NO: 22, and a light chainvariable region that is at least 95%, 96%, 97%, 98%, or 99% identical tothe sequence of SEQ ID NO: 23. In a further embodiment, the antigenbinding moiety, particularly Fab molecule, which specifically binds toCEA comprises the heavy chain variable region sequence of SEQ ID NO: 22,and the light chain variable region sequence of SEQ ID NO: 23. In oneembodiment, the antibody is a Fab molecule.

Polynucleotides

The invention further provides isolated polynucleotides encoding a Tcell activating bispecific antigen binding molecule as described hereinor a fragment thereof. In some embodiments, said fragment is an antigenbinding fragment.

The polynucleotides encoding T cell activating bispecific antigenbinding molecules of the invention may be expressed as a singlepolynucleotide that encodes the entire T cell activating bispecificantigen binding molecule or as multiple (e.g., two or more)polynucleotides that are co-expressed. Polypeptides encoded bypolynucleotides that are co-expressed may associate through, e.g.,disulfide bonds or other means to form a functional T cell activatingbispecific antigen binding molecule. For example, the light chainportion of a Fab molecule may be encoded by a separate polynucleotidefrom the portion of the T cell activating bispecific antigen bindingmolecule comprising the heavy chain portion of the Fab molecule, an Fcdomain subunit and optionally (part of) another Fab molecule. Whenco-expressed, the heavy chain polypeptides will associate with the lightchain polypeptides to form the Fab molecule. In another example, theportion of the T cell activating bispecific antigen binding moleculecomprising one of the two Fc domain subunits and optionally (part of)one or more Fab molecules could be encoded by a separate polynucleotidefrom the portion of the T cell activating bispecific antigen bindingmolecule comprising the the other of the two Fc domain subunits andoptionally (part of) a Fab molecule. When co-expressed, the Fc domainsubunits will associate to form the Fc domain.

In some embodiments, the isolated polynucleotide encodes the entire Tcell activating bispecific antigen binding molecule according to theinvention as described herein. In other embodiments, the isolatedpolynucleotide encodes a polypeptides comprised in the T cell activatingbispecific antigen binding molecule according to the invention asdescribed herein.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

T cell activating bispecific antigen binding molecules of the inventionmay be obtained, for example, by solid-state peptide synthesis (e.g.Merrifield solid phase synthesis) or recombinant production. Forrecombinant production one or more polynucleotide encoding the T cellactivating bispecific antigen binding molecule (fragment), e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such polynucleotidemay be readily isolated and sequenced using conventional procedures. Inone embodiment a vector, preferably an expression vector, comprising oneor more of the polynucleotides of the invention is provided. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of a T cellactivating bispecific antigen binding molecule (fragment) along withappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, N.Y (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe T cell activating bispecific antigen binding molecule (fragment)(i.e. the coding region) is cloned in operable association with apromoter and/or other transcription or translation control elements. Asused herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, if present, but any flankingsequences, for example promoters, ribosome binding sites,transcriptional terminators, introns, 5′ and 3′ untranslated regions,and the like, are not part of a coding region. Two or more codingregions can be present in a single polynucleotide construct, e.g. on asingle vector, or in separate polynucleotide constructs, e.g. onseparate (different) vectors. Furthermore, any vector may contain asingle coding region, or may comprise two or more coding regions, e.g. avector of the present invention may encode one or more polypeptides,which are post- or co-translationally separated into the final proteinsvia proteolytic cleavage. In addition, a vector, polynucleotide, ornucleic acid of the invention may encode heterologous coding regions,either fused or unfused to a polynucleotide encoding the T cellactivating bispecific antigen binding molecule (fragment) of theinvention, or variant or derivative thereof. Heterologous coding regionsinclude without limitation specialized elements or motifs, such as asecretory signal peptide or a heterologous functional domain. Anoperable association is when a coding region for a gene product, e.g. apolypeptide, is associated with one or more regulatory sequences in sucha way as to place expression of the gene product under the influence orcontrol of the regulatory sequence(s). Two DNA fragments (such as apolypeptide coding region and a promoter associated therewith) are“operably associated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein. A variety of transcription controlregions are known to those skilled in the art. These include, withoutlimitation, transcription control regions, which function in vertebratecells, such as, but not limited to, promoter and enhancer segments fromcytomegaloviruses (e.g. the immediate early promoter, in conjunctionwith intron-A), simian virus 40 (e.g. the early promoter), andretroviruses (such as, e.g. Rous sarcoma virus). Other transcriptioncontrol regions include those derived from vertebrate genes such asactin, heat shock protein, bovine growth hormone and rabbit a-globin, aswell as other sequences capable of controlling gene expression ineukaryotic cells. Additional suitable transcription control regionsinclude tissue-specific promoters and enhancers as well as induciblepromoters (e.g. promoters inducible tetracyclins). Similarly, a varietyof translation control elements are known to those of ordinary skill inthe art. These include, but are not limited to ribosome binding sites,translation initiation and termination codons, and elements derived fromviral systems (particularly an internal ribosome entry site, or IRES,also referred to as a CITE sequence). The expression cassette may alsoinclude other features such as an origin of replication, and/orchromosome integration elements such as retroviral long terminal repeats(LTRs), or adeno-associated viral (AAV) inverted terminal repeats(ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the T cell activating bispecific antigen binding molecule is desired,DNA encoding a signal sequence may be placed upstream of the nucleicacid encoding a T cell activating bispecific antigen binding molecule ofthe invention or a fragment thereof. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide.

In certain embodiments, the native signal peptide, e.g. animmunoglobulin heavy chain or light chain signal peptide is used, or afunctional derivative of that sequence that retains the ability todirect the secretion of the polypeptide that is operably associated withit. Alternatively, a heterologous mammalian signal peptide, or afunctional derivative thereof, may be used. For example, the wild-typeleader sequence may be substituted with the leader sequence of humantissue plasminogen activator (TPA) or mouse 0-glucuronidase.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling the Tcell activating bispecific antigen binding molecule may be includedwithin or at the ends of the T cell activating bispecific antigenbinding molecule (fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes (part of) a T cell activatingbispecific antigen binding molecule of the invention. As used herein,the term “host cell” refers to any kind of cellular system which can beengineered to generate the T cell activating bispecific antigen bindingmolecules of the invention or fragments thereof. Host cells suitable forreplicating and for supporting expression of T cell activatingbispecific antigen binding molecules are well known in the art. Suchcells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vector containingcells can be grown for seeding large scale fermenters to obtainsufficient quantities of the T cell activating bispecific antigenbinding molecule for clinical applications. Suitable host cells includeprokaryotic microorganisms, such as E. coli, or various eukaryoticcells, such as Chinese hamster ovary cells (CHO), insect cells, or thelike. For example, polypeptides may be produced in bacteria inparticular when glycosylation is not needed. After expression, thepolypeptide may be isolated from the bacterial cell paste in a solublefraction and can be further purified. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are suitablecloning or expression hosts for polypeptide-encoding vectors, includingfungi and yeast strains whose glycosylation pathways have been“humanized”, resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006). Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cells includeplant and insect cells. Numerous baculoviral strains have beenidentified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts. See e.g. U.S. Pat. Nos.5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describingPLANTIBODIES® technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)),baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkeykidney cells (CV1), African green monkey kidney cells (VERO-76), humancervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo ratliver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT 060562), TRI cells (as described,e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5cells, and FS4 cells. Other useful mammalian host cell lines includeChinese hamster ovary (CHO) cells, including dhfr⁻ CHO cells (Urlaub etal., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell linessuch as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian hostcell lines suitable for protein production, see, e.g., Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells,e.g., mammalian cultured cells, yeast cells, insect cells, bacterialcells and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant or animaltissue. In one embodiment, the host cell is a eukaryotic cell,preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell,a human embryonic kidney (HEK) cell or a lymphoid cell (e.g., YO, NS0,Sp20 cell).

Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a polypeptide comprising either theheavy or the light chain of an antigen binding domain such as anantibody, may be engineered so as to also express the other of theantibody chains such that the expressed product is an antibody that hasboth a heavy and a light chain.

In one embodiment, a method of producing a T cell activating bispecificantigen binding molecule according to the invention is provided, whereinthe method comprises culturing a host cell comprising a polynucleotideencoding the T cell activating bispecific antigen binding molecule, asprovided herein, under conditions suitable for expression of the T cellactivating bispecific antigen binding molecule, and recovering the Tcell activating bispecific antigen binding molecule from the host cell(or host cell culture medium).

The components of the T cell activating bispecific antigen bindingmolecule are genetically fused to each other. T cell activatingbispecific antigen binding molecule can be designed such that itscomponents are fused directly to each other or indirectly through alinker sequence. The composition and length of the linker may bedetermined in accordance with methods well known in the art and may betested for efficacy. Examples of linker sequences between differentcomponents of T cell activating bispecific antigen binding molecules arefound in the sequences provided herein. Additional sequences may also beincluded to incorporate a cleavage site to separate the individualcomponents of the fusion if desired, for example an endopeptidaserecognition sequence.

In certain embodiments the one or more antigen binding moieties of the Tcell activating bispecific antigen binding molecules comprise at leastan antibody variable region capable of binding an antigenic determinant.Variable regions can form part of and be derived from naturally ornon-naturally occurring antibodies and fragments thereof. Methods toproduce polyclonal antibodies and monoclonal antibodies are well knownin the art (see e.g. Harlow and Lane, “Antibodies, a laboratory manual”,Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodiescan be constructed using solid phase-peptide synthesis, can be producedrecombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or can beobtained, for example, by screening combinatorial libraries comprisingvariable heavy chains and variable light chains (see e.g. U.S. Pat. No.5,969,108 to McCafferty).

Any animal species of antibody, antibody fragment, antigen bindingdomain or variable region can be used in the T cell activatingbispecific antigen binding molecules of the invention. Non-limitingantibodies, antibody fragments, antigen binding domains or variableregions useful in the present invention can be of murine, primate, orhuman origin. If the T cell activating bispecific antigen bindingmolecule is intended for human use, a chimeric form of antibody may beused wherein the constant regions of the antibody are from a human. Ahumanized or fully human form of the antibody can also be prepared inaccordance with methods well known in the art (see e.g. U.S. Pat. No.5,565,332 to Winter). Humanization may be achieved by various methodsincluding, but not limited to (a) grafting the non-human (e.g., donorantibody) CDRs onto human (e.g. recipient antibody) framework andconstant regions with or without retention of critical frameworkresidues (e.g. those that are important for retaining good antigenbinding affinity or antibody functions), (b) grafting only the non-humanspecificity-determining regions (SDRs or a-CDRs; the residues criticalfor the antibody-antigen interaction) onto human framework and constantregions, or (c) transplanting the entire non-human variable domains, but“cloaking” them with a human-like section by replacement of surfaceresidues. Humanized antibodies and methods of making them are reviewed,e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), andare further described, e.g., in Riechmann et al., Nature 332, 323-329(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989);U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones etal., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81,6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988);Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005)(describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498(1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36, 43-60(2005) (describing “FR shuffling”); and Osbourn et al., Methods 36,61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000)(describing the “guided selection” approach to FR shuffling). Humanantibodies and human variable regions can be produced using varioustechniques known in the art. Human antibodies are described generally invan Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) andLonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regionscan form part of and be derived from human monoclonal antibodies made bythe hybridoma method (see e.g. Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Human antibodies and human variable regions may also be prepared byadministering an immunogen to a transgenic animal that has been modifiedto produce intact human antibodies or intact antibodies with humanvariable regions in response to antigenic challenge (see e.g. Lonberg,Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variableregions may also be generated by isolating Fv clone variable regionsequences selected from human-derived phage display libraries (see e.g.,Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001); and McCafferty et al.,Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phagetypically display antibody fragments, either as single-chain Fv (scFv)fragments or as Fab fragments.

In certain embodiments, the antigen binding moieties useful in thepresent invention are engineered to have enhanced binding affinityaccording to, for example, the methods disclosed in U.S. Pat. Appl.Publ. No. 2004/0132066, the entire contents of which are herebyincorporated by reference. The ability of the T cell activatingbispecific antigen binding molecule of the invention to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance technique(analyzed on a BIACORE® T100 system) (Liljeblad, et al., Glyco J 17,323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)). Competition assays may be used to identify an antibody,antibody fragment, antigen binding domain or variable domain thatcompetes with a reference antibody for binding to a particular antigen,e.g. an antibody that competes with the V9 antibody for binding to CD3.In certain embodiments, such a competing antibody binds to the sameepitope (e.g. a linear or a conformational epitope) that is bound by thereference antibody. Detailed exemplary methods for mapping an epitope towhich an antibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.). In an exemplary competition assay, immobilized antigen(e.g. CD3) is incubated in a solution comprising a first labeledantibody that binds to the antigen (e.g. V9 antibody, described in U.S.Pat. No. 6,054,297) and a second unlabeled antibody that is being testedfor its ability to compete with the first antibody for binding to theantigen. The second antibody may be present in a hybridoma supernatant.As a control, immobilized antigen is incubated in a solution comprisingthe first labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto the antigen, excess unbound antibody is removed, and the amount oflabel associated with immobilized antigen is measured. If the amount oflabel associated with immobilized antigen is substantially reduced inthe test sample relative to the control sample, then that indicates thatthe second antibody is competing with the first antibody for binding tothe antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manualch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

T cell activating bispecific antigen binding molecules prepared asdescribed herein may be purified by art-known techniques such as highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, affinity chromatography, size exclusion chromatography,and the like. The actual conditions used to purify a particular proteinwill depend, in part, on factors such as net charge, hydrophobicity,hydrophilicity etc., and will be apparent to those having skill in theart. For affinity chromatography purification an antibody, ligand,receptor or antigen can be used to which the T cell activatingbispecific antigen binding molecule binds. For example, for affinitychromatography purification of T cell activating bispecific antigenbinding molecules of the invention, a matrix with protein A or protein Gmay be used. Sequential Protein A or G affinity chromatography and sizeexclusion chromatography can be used to isolate a T cell activatingbispecific antigen binding molecule essentially as described in theExamples. The purity of the T cell activating bispecific antigen bindingmolecule can be determined by any of a variety of well known analyticalmethods including gel electrophoresis, high pressure liquidchromatography, and the like. For example, the heavy chain fusionproteins expressed as described in the Examples were shown to be intactand properly assembled as demonstrated by reducing SDS-PAGE (see e.g.FIG. 4). Three bands were resolved at approximately Mr 25,000, Mr 50,000and Mr 75,000, corresponding to the predicted molecular weights of the Tcell activating bispecific antigen binding molecule light chain, heavychain and heavy chain/light chain fusion protein.

Assays

T cell activating bispecific antigen binding molecules provided hereinmay be identified, screened for, or characterized for theirphysical/chemical properties and/or biological activities by variousassays known in the art.

Affinity Assays

The affinity of the T cell activating bispecific antigen bindingmolecule for an Fc receptor or a target antigen can be determined inaccordance with the methods set forth in the Examples by surface plasmonresonance (SPR), using standard instrumentation such as a BIACORE®instrument (GE Healthcare), and receptors or target proteins such as maybe obtained by recombinant expression. Alternatively, binding of T cellactivating bispecific antigen binding molecules for different receptorsor target antigens may be evaluated using cell lines expressing theparticular receptor or target antigen, for example by flow cytometry(FACS). A specific illustrative and exemplary embodiment for measuringbinding affinity is described in the following and in the Examplesbelow. According to one embodiment, K_(D) is measured by surface plasmonresonance using a BIACORE® T100 machine (GE Healthcare) at 25° C.

To analyze the interaction between the Fc-portion and Fc receptors,His-tagged recombinant Fc-receptor is captured by an anti-Penta Hisantibody (Qiagen) immobilized on CM5 chips and the bispecific constructsare used as analytes. Briefly, carboxymethylated dextran biosensor chips(CM5, GE Healthcare) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Anti Penta-His antibody is diluted with 10 mM sodium acetate, pH 5.0, to40 g/ml before injection at a flow rate of 5 μl/min to achieveapproximately 6500 response units (RU) of coupled protein. Following theinjection of the ligand, 1 M ethanolamine is injected to block unreactedgroups. Subsequently the Fc-receptor is captured for 60 s at 4 or 10 nM.For kinetic measurements, four-fold serial dilutions of the bispecificconstruct (range between 500 nM and 4000 nM) are injected in HBS-EP (GEHealthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20,pH 7.4) at 25° C. at a flow rate of 30 μl/min for 120 s.

To determine the affinity to the target antigen, bispecific constructsare captured by an anti human Fab specific antibody (GE Healthcare) thatis immobilized on an activated CM5-sensor chip surface as described forthe anti Penta-His antibody. The final amount of coupled protein is isapproximately 12000 R U. The bispecific constructs are captured for 90 sat 300 nM. The target antigens are passed through the flow cells for 180s at a concentration range from 250 to 1000 nM with a flowrate of 30μl/min. The dissociation is monitored for 180 s.

Bulk refractive index differences are corrected for by subtracting theresponse obtained on reference flow cell. The steady state response wasused to derive the dissociation constant K_(D) by non-linear curvefitting of the Langmuir binding isotherm. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® T100 Evaluation Software version 1.1.1)by simultaneously fitting the association and dissociation sensorgrams.The equilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

Activity Assays

Biological activity of the T cell activating bispecific antigen bindingmolecules of the invention can be measured by various assays asdescribed in the Examples. Biological activities may for example includethe induction of proliferation of T cells, the induction of signaling inT cells, the induction of expression of activation markers in T cells,the induction of cytokine secretion by T cells, the induction of lysisof target cells such as tumor cells, and the induction of tumorregression and/or the improvement of survival.

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the T cell activating bispecific antigen bindingmolecules provided herein, e.g., for use in any of the below therapeuticmethods. In one embodiment, a pharmaceutical composition comprises anyof the T cell activating bispecific antigen binding molecules providedherein and a pharmaceutically acceptable carrier. In another embodiment,a pharmaceutical composition comprises any of the T cell activatingbispecific antigen binding molecules provided herein and at least oneadditional therapeutic agent, e.g., as described below.

Further provided is a method of producing a T cell activating bispecificantigen binding molecule of the invention in a form suitable foradministration in vivo, the method comprising (a) obtaining a T cellactivating bispecific antigen binding molecule according to theinvention, and (b) formulating the T cell activating bispecific antigenbinding molecule with at least one pharmaceutically acceptable carrier,whereby a preparation of T cell activating bispecific antigen bindingmolecule is formulated for administration in vivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more T cell activatingbispecific antigen binding molecule dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that are generally non-toxic to recipients at the dosagesand concentrations employed, i.e. do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of a pharmaceuticalcomposition that contains at least one T cell activating bispecificantigen binding molecule and optionally an additional active ingredientwill be known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference.Moreover, for animal (e.g., human) administration, it will be understoodthat preparations should meet sterility, pyrogenicity, general safetyand purity standards as required by FDA Office of Biological Standardsor corresponding authorities in other countries. Preferred compositionsare lyophilized formulations or aqueous solutions. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,buffers, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g. antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, antioxidants,proteins, drugs, drug stabilizers, polymers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. T cell activating bispecific antigen binding molecules of thepresent invention (and any additional therapeutic agent) can beadministered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrasplenically, intrarenally, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, intratumorally, intramuscularly, intraperitoneally,subcutaneously, subconjunctivally, intravesicularlly, mucosally,intrapericardially, intraumbilically, intraocularally, orally,topically, locally, by inhalation (e.g. aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g. liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference). Parenteral administration, inparticular intravenous injection, is most commonly used foradministering polypeptide molecules such as the T cell activatingbispecific antigen binding molecules of the invention.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the T cell activating bispecific antigen bindingmolecules of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the T cell activatingbispecific antigen binding molecules may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. Sterile injectable solutions are prepared by incorporatingthe T cell activating bispecific antigen binding molecules of theinvention in the required amount in the appropriate solvent with variousof the other ingredients enumerated below, as required. Sterility may bereadily accomplished, e.g., by filtration through sterile filtrationmembranes. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and/or the other ingredients. Inthe case of sterile powders for the preparation of sterile injectablesolutions, suspensions or emulsion, the preferred methods of preparationare vacuum-drying or freeze-drying techniques which yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered liquid medium thereof. The liquid mediumshould be suitably buffered if necessary and the liquid diluent firstrendered isotonic prior to injection with sufficient saline or glucose.The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable carriers include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, the T cellactivating bispecific antigen binding molecules may also be formulatedas a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, the Tcell activating bispecific antigen binding molecules may be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the T cell activating bispecificantigen binding molecules of the invention may be manufactured by meansof conventional mixing, dissolving, emulsifying, encapsulating,entrapping or lyophilizing processes. Pharmaceutical compositions may beformulated in conventional manner using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries whichfacilitate processing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The T cell activating bispecific antigen binding molecules may beformulated into a composition in a free acid or base, neutral or saltform. Pharmaceutically acceptable salts are salts that substantiallyretain the biological activity of the free acid or base. These includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Pharmaceutical salts tend to be more soluble inaqueous and other protic solvents than are the corresponding free baseforms.

Therapeutic Methods and Compositions

Any of the T cell activating bispecific antigen binding moleculesprovided herein may be used in therapeutic methods. T cell activatingbispecific antigen binding molecules of the invention can be used asimmunotherapeutic agents, for example in the treatment of cancers.

For use in therapeutic methods, T cell activating bispecific antigenbinding molecules of the invention would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

In one aspect, T cell activating bispecific antigen binding molecules ofthe invention for use as a medicament are provided. In further aspects,T cell activating bispecific antigen binding molecules of the inventionfor use in treating a disease are provided. In certain embodiments, Tcell activating bispecific antigen binding molecules of the inventionfor use in a method of treatment are provided. In one embodiment, theinvention provides a T cell activating bispecific antigen bindingmolecule as described herein for use in the treatment of a disease in anindividual in need thereof. In certain embodiments, the inventionprovides a T cell activating bispecific antigen binding molecule for usein a method of treating an individual having a disease comprisingadministering to the individual a therapeutically effective amount ofthe T cell activating bispecific antigen binding molecule. In certainembodiments the disease to be treated is a proliferative disorder. In aparticular embodiment the disease is cancer. In certain embodiments themethod further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated iscancer. In further embodiments, the invention provides a T cellactivating bispecific antigen binding molecule as described herein foruse in inducing lysis of a target cell, particularly a tumor cell. Incertain embodiments, the invention provides a T cell activatingbispecific antigen binding molecule for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of the Tcell activating bispecific antigen binding molecule to induce lysis of atarget cell. An “individual” according to any of the above embodimentsis a mammal, preferably a human.

In a further aspect, the invention provides for the use of a T cellactivating bispecific antigen binding molecule of the invention in themanufacture or preparation of a medicament. In one embodiment themedicament is for the treatment of a disease in an individual in needthereof. In a further embodiment, the medicament is for use in a methodof treating a disease comprising administering to an individual havingthe disease a therapeutically effective amount of the medicament. Incertain embodiments the disease to be treated is a proliferativedisorder. In a particular embodiment the disease is cancer. In oneembodiment, the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In a further embodiment, the medicament is forinducing lysis of a target cell, particularly a tumor cell. In still afurther embodiment, the medicament is for use in a method of inducinglysis of a target cell, particularly a tumor cell, in an individualcomprising administering to the individual an effective amount of themedicament to induce lysis of a target cell. An “individual” accordingto any of the above embodiments may be a mammal, preferably a human.

In a further aspect, the invention provides a method for treating adisease. In one embodiment, the method comprises administering to anindividual having such disease a therapeutically effective amount of a Tcell activating bispecific antigen binding molecule of the invention. Inone embodiment a composition is administered to said individual,comprising the T cell activating bispecific antigen binding molecule ofthe invention in a pharmaceutically acceptable form. In certainembodiments the disease to be treated is a proliferative disorder. In aparticular embodiment the disease is cancer. In certain embodiments themethod further comprises administering to the individual atherapeutically effective amount of at least one additional therapeuticagent, e.g., an anti-cancer agent if the disease to be treated iscancer. An “individual” according to any of the above embodiments may bea mammal, preferably a human.

In a further aspect, the invention provides a method for inducing lysisof a target cell, particularly a tumor cell. In one embodiment themethod comprises contacting a target cell with a T cell activatingbispecific antigen binding molecule of the invention in the presence ofa T cell, particularly a cytotoxic T cell. In a further aspect, a methodfor inducing lysis of a target cell, particularly a tumor cell, in anindividual is provided. In one such embodiment, the method comprisesadministering to the individual an effective amount of a T cellactivating bispecific antigen binding molecule to induce lysis of atarget cell. In one embodiment, an “individual” is a human.

In certain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Non-limiting examples of cancers includebladder cancer, brain cancer, head and neck cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, blood cancer,skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferation disorders that can be treated using a T cellactivating bispecific antigen binding molecule of the present inventioninclude, but are not limited to neoplasms located in the: abdomen, bone,breast, digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvic, skin, soft tissue, spleen, thoracic region, andurogenital system. Also included are pre-cancerous conditions or lesionsand cancer metastases. In certain embodiments the cancer is chosen fromthe group consisting of renal cell cancer, skin cancer, lung cancer,colorectal cancer, breast cancer, brain cancer, head and neck cancer,gastric cancer, pancreatic cancer, ovarian cancer. In one embodiment,the cancer is a solid tumor. A skilled artisan readily recognizes thatin many cases the T cell activating bispecific antigen binding moleculemay not provide a cure but may only provide partial benefit. In someembodiments, a physiological change having some benefit is alsoconsidered therapeutically beneficial. Thus, in some embodiments, anamount of T cell activating bispecific antigen binding molecule thatprovides a physiological change is considered an “effective amount” or a“therapeutically effective amount”. The subject, patient, or individualin need of treatment is typically a mammal, more specifically a human.

In some embodiments, an effective amount of a T cell activatingbispecific antigen binding molecule of the invention is administered toa cell. In other embodiments, a therapeutically effective amount of a Tcell activating bispecific antigen binding molecule of the invention isadministered to an individual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of aT cell activating bispecific antigen binding molecule of the invention(when used alone or in combination with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the route of administration, the body weight of the patient, the type ofT cell activating bispecific antigen binding molecule, the severity andcourse of the disease, whether the T cell activating bispecific antigenbinding molecule is administered for preventive or therapeutic purposes,previous or concurrent therapeutic interventions, the patient's clinicalhistory and response to the T cell activating bispecific antigen bindingmolecule, and the discretion of the attending physician. Thepractitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject. Various dosing schedulesincluding but not limited to single or multiple administrations overvarious time-points, bolus administration, and pulse infusion arecontemplated herein.

The T cell activating bispecific antigen binding molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of T cell activating bispecific antigenbinding molecule can be an initial candidate dosage for administrationto the patient, whether, for example, by one or more separateadministrations, or by continuous infusion. One typical daily dosagemight range from about 1 μg/kg to 100 mg/kg or more, depending on thefactors mentioned above. For repeated administrations over several daysor longer, depending on the condition, the treatment would generally besustained until a desired suppression of disease symptoms occurs. Oneexemplary dosage of the T cell activating bispecific antigen bindingmolecule would be in the range from about 0.005 mg/kg to about 10 mg/kg.In other non-limiting examples, a dose may also comprise from about 1microgram/kg body weight, about 5 microgram/kg body weight, about 10microgram/kg body weight, about 50 microgram/kg body weight, about 100microgram/kg body weight, about 200 microgram/kg body weight, about 350microgram/kg body weight, about 500 microgram/kg body weight, about 1milligram/kg body weight, about 5 milligram/kg body weight, about 10milligram/kg body weight, about 50 milligram/kg body weight, about 100milligram/kg body weight, about 200 milligram/kg body weight, about 350milligram/kg body weight, about 500 milligram/kg body weight, to about1000 mg/kg body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg body weight to about100 mg/kg body weight, about 5 microgram/kg body weight to about 500milligram/kg body weight, etc., can be administered, based on thenumbers described above. Thus, one or more doses of about 0.5 mg/kg, 2.0mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may beadministered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the T cell activating bispecific antigen binding molecule). Aninitial higher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The T cell activating bispecific antigen binding molecules of theinvention will generally be used in an amount effective to achieve theintended purpose. For use to treat or prevent a disease condition, the Tcell activating bispecific antigen binding molecules of the invention,or pharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the T cell activating bispecific antigen bindingmolecules which are sufficient to maintain therapeutic effect. Usualpatient dosages for administration by injection range from about 0.1 to50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeuticallyeffective plasma levels may be achieved by administering multiple doseseach day. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the T cell activating bispecific antigen bindingmolecules may not be related to plasma concentration. One having skillin the art will be able to optimize therapeutically effective localdosages without undue experimentation.

A therapeutically effective dose of the T cell activating bispecificantigen binding molecules described herein will generally providetherapeutic benefit without causing substantial toxicity. Toxicity andtherapeutic efficacy of a T cell activating bispecific antigen bindingmolecule can be determined by standard pharmaceutical procedures in cellculture or experimental animals. Cell culture assays and animal studiescan be used to determine the LD₅₀ (the dose lethal to 50% of apopulation) and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. T cellactivating bispecific antigen binding molecules that exhibit largetherapeutic indices are preferred. In one embodiment, the T cellactivating bispecific antigen binding molecule according to the presentinvention exhibits a high therapeutic index. The data obtained from cellculture assays and animal studies can be used in formulating a range ofdosages suitable for use in humans. The dosage lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage may vary within this range depending upon avariety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety). Theattending physician for patients treated with T cell activatingbispecific antigen binding molecules of the invention would know how andwhen to terminate, interrupt, or adjust administration due to toxicity,organ dysfunction, and the like. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministered dose in the management of the disorder of interest willvary with the severity of the condition to be treated, with the route ofadministration, and the like. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency will also varyaccording to the age, body weight, and response of the individualpatient.

Other Agents and Treatments

The T cell activating bispecific antigen binding molecules of theinvention may be administered in combination with one or more otheragents in therapy. For instance, a T cell activating bispecific antigenbinding molecule of the invention may be co-administered with at leastone additional therapeutic agent. The term “therapeutic agent”encompasses any agent administered to treat a symptom or disease in anindividual in need of such treatment. Such additional therapeutic agentmay comprise any active ingredients suitable for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. In certain embodiments, anadditional therapeutic agent is an immunomodulatory agent, a cytostaticagent, an inhibitor of cell adhesion, a cytotoxic agent, an activator ofcell apoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers. In a particular embodiment, the additionaltherapeutic agent is an anti-cancer agent, for example a microtubuledisruptor, an antimetabolite, a topoisomerase inhibitor, a DNAintercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of T cell activating bispecificantigen binding molecule used, the type of disorder or treatment, andother factors discussed above. The T cell activating bispecific antigenbinding molecules are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the T cell activating bispecific antigen bindingmolecule of the invention can occur prior to, simultaneously, and/orfollowing, administration of the additional therapeutic agent and/oradjuvant. T cell activating bispecific antigen binding molecules of theinvention can also be used in combination with radiation therapy.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a T cell activating bispecific antigen binding moleculeof the invention. The label or package insert indicates that thecomposition is used for treating the condition of choice. Moreover, thearticle of manufacture may comprise (a) a first container with acomposition contained therein, wherein the composition comprises a Tcell activating bispecific antigen binding molecule of the invention;and (b) a second container with a composition contained therein, whereinthe composition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

General Methods Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturers'instructions. General information regarding the nucleotide sequences ofhuman immunoglobulins light and heavy chains is given in: Kabat, E. A.et al., (1991) Sequences of Proteins of Immunological Interest, 5^(th)ed., NIH Publication No. 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments where required were either generated by PCR usingappropriate templates or were synthesized by Geneart AG (Regensburg,Germany) from synthetic oligonucleotides and PCR products by automatedgene synthesis. In cases where no exact gene sequence was available,oligonucleotide primers were designed based on sequences from closesthomologues and the genes were isolated by RT-PCR from RNA originatingfrom the appropriate tissue. The gene segments flanked by singularrestriction endonuclease cleavage sites were cloned into standardcloning/sequencing vectors. The plasmid DNA was purified fromtransformed bacteria and concentration determined by UV spectroscopy.The DNA sequence of the subcloned gene fragments was confirmed by DNAsequencing. Gene segments were designed with suitable restriction sitesto allow sub-cloning into the respective expression vectors. Allconstructs were designed with a 5′-end DNA sequence coding for a leaderpeptide which targets proteins for secretion in eukaryotic cells.

Example 1 Binding of Different Humanized Variants of T84.66 IgG to Cells

Novel humanized variants of the murine antibody T84.66 (Wagener et al.,J Immunol 130, 2308 (1983), Neumaier et al., J Immunol 135, 3604 (1985))were developed by grafting of the CDRs onto human germline frameworkacceptor sequences.

In this example, the binding of different humanized variants of T84.66IgG was tested on CEA-expressing human gastric adenocarcinoma cells(MKN45, DSMZ ACC 409).

Briefly, cells were harvested, counted, checked for viability andre-suspended at 2×10⁶ cells/ml in FACS buffer (100 μl PBS 0.1% BSA). 100μl of cell suspension (containing 0.2×10⁶ cells) were incubated inround-bottom 96-well plate for 30 min at 4° C. with increasingconcentrations of the CEA IgG (4 ng/ml-60 g/ml), washed twice with coldPBS 0.1% BSA, re-incubated for further 30 min at 4° C. with thePE-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgG FcgFragment Specific secondary antibody (Jackson Immuno Research Lab PE#109-116-170), washed twice with cold PBS 0.1% BSA and immediatelyanalyzed by FACS using a FACS CantoII (Software FACS Diva). Bindingcurves and EC50 values were obtained and calculated using GraphPadPrism5(FIG. 2, binding to MKN45 cells).

FIG. 2 shows the different binding pattern of selected humanizedvariants of the T84.66 IgG to human CEA, expressed on MKN45 cells. Basedon the binding pattern and the calculated EC50 binding values (Table 1),the humanized variant 1 (SEQ ID NOs 22 and 23) was selected for furtherevaluation.

TABLE 1 Binding of different humanized variants of T84.66 IgGs to cells(EC50 values, based on binding curves shown in FIG. 2, calculated byGraph Pad Prism). EC50 (μg/ml) Parental chimeric T84.66 0.99 Humanizedvariant 1 1.5 Humanized variant 2 8.6 Humanized variant 3 1.4 Humanizedvariant 4 3.1 Humanized variant 5 — Humanized variant 6 —

Example 2 Preparation of Anti-CEA/Anti-CD3 T Cell Bispecific (TCB)Molecules

The following molecules were prepared in this example; schematicillustrations thereof are shown in FIG. 3:

-   -   A. “2+1 IgG CrossFab, inverted” with charge modifications (VH/VL        exchange in CD3 binder, charge modification in CEA binder,        parental murine CEA binder (T84.66)) (FIG. 3A, SEQ ID NOs 32-35)    -   B. “2+1 IgG CrossFab, inverted” with charge modifications (VH/VL        exchange in CD3 binder, charge modification in CEA binder,        humanized CEA binder) (FIG. 3B, SEQ ID NOs 34, 36-38)    -   C. “1+1 IgG CrossFab, inverted” with charge modifications (VH/VL        exchange in CD3 binder, charge modification in CEA binder,        humanized CEA binder) (FIG. 3C, SEQ ID NOs 34, 37-39)    -   D. “1+1 IgG CrossMab” with charge modifications (VH/VL exchange        in CD3 binder, charge modification in CEA binder, humanized CEA        binder) (FIG. 3D, SEQ ID NOs 34, 36, 38, 40)    -   E. “2+1 IgG CrossFab, inverted” with charge modifications (VH/VL        exchange in CD3 binder, charge modification in CEA binder,        humanized CEA binder, longer linker) (FIG. 3E, SEQ ID NOs 34,        36, 38, 41).    -   F. “2+1 IgG CrossFab, inverted” without charge modifications        (VH/VL exchange in CD3 binder, humanized CEA binder) (FIG. 3F,        SEQ ID NOs 34, 50-52).

The DNA sequences encoding the variable heavy and light chain regions ofthe CD3 and CEA binders were subcloned in frame with the respectiveconstant regions which are pre-inserted into the respective recipientmammalian expression vector. Protein expression is driven by an MPSV ora CMV promoter. Polyadenylation is driven by a synthetic polyA signalsequence located at the 3′ end of the CDS. In addition each vectorcontains an EBV OriP sequence for autosomal replication.

For production of the molecules, HEK293-EBNA cells growing in suspensionwere co-transfected with the respective expression vectors usingpolyethylenimine (PEI) as transfection reagent. The cells weretransfected with the corresponding expression vectors in a 1:2:1:1 ratio(A, B, E and F: “vector heavy chain (VH-CH1-VL-CH1-CH2-CH3)”: “vectorlight chain (VL-CL)”: “vector heavy chain (VH-CH1-CH2-CH3)”: “vectorlight chain (VH-CL)”) or in a 1:1:1:1 ratio (C: “vector heavy chain(VH-CH1-VL-CH1-CH2-CH3)”: “vector light chain (VL-CL)”: “vector heavychain (CH2-CH3)”: “vector light chain (VH-CL)”, D: “vector heavy chain(VL-CH1-CH2-CH3)”: “vector light chain (VL-CL)”: “vector heavy chain(VH-CH1-CH2-CH3)”: “vector light chain (VH-CL)”).

For transfection, HEK293 EBNA cells were cultivated in suspension serumfree in Excell culture medium containing 6 mM L-glutamine and 250 mg/lG418. For the production in 600 ml tubespin flasks (max. working volume400 ml) 600 million HEK293 EBNA cells were seeded 24 hours beforetransfection. For transfection, cells were centrifuged for 5 min at210×g, and supernatant was replaced by 20 ml pre-warmed CD CHO medium.Expression vectors are mixed in 20 ml CD CHO medium to a final amount of400 μg DNA. After addition of 1080 μl PEI solution (2.7 μg/ml) themixture was vortexed for 15 s and subsequently incubated for 10 min atroom temperature. Afterwards cells were mixed with the DNA/PEI solution,transferred to a 600 ml tubespin flask and incubated for 3 hours at 37°C. in an incubator with a humidified 5% CO₂ atmosphere. Afterincubation, 360 ml Excell medium containing 6 mM L-glutamine, 5 g/LPepsoy and 1.0 mM VPA was added and cells were cultivated for 24 hours.One day after transfection 7% Feed 1 was added. After 7 days cultivationsupernatant was collected for purification by centrifugation for 20-30min at 3600×g (Sigma 8K centrifuge), the solution was sterile filtered(0.22 μm filter) and sodium azide in a final concentration of 0.01% w/vwas added. The solution was kept at 4° C.

The titer of the molecules in the culture medium was determined byProtein A-HPLC (Table 2). Calculation of the titer is based on atwo-step process and includes binding of Fc-containing molecules toProtein A at pH 8.0 and release in a step elution at pH 2.5. Bothbuffers used for the analysis contained Tris (10 mM), glycine (50 mM),and NaCl (100 mM) and were adjusted to the respective pHs (8 and 2.5).The column body was an Upchurch 2×20 mm pre-column with an internalvolume of ˜63 μl packed with POROS 20A. After initial calibration, 100μl of each sample was injected with a flow rate of 0.5 ml/min. After0.67 minutes the sample was eluted with a pH step to pH 2.5.Quantitation was done by determination of 280 nm absorbance andcalculation using a standard curve with a concentration range of humanIgG1 from 16 to 166 mg/l.

The secreted proteins were purified from cell culture supernatants byaffinity chromatography using Protein A affinity chromatography,followed by a size exclusion chromatographic step.

For affinity chromatography supernatant was loaded on a HITRAP® ProteinAHP (molecule A, B and F) or a MabSelectSure (molecule C, D and E) column(CV=5 mL, GE Healthcare) equilibrated with 25 ml 20 mM sodium phosphate,20 mM sodium citrate, pH 7.5. Unbound protein was removed by washingwith at least 10 column volumes 20 mM sodium phosphate, 20 mM sodiumcitrate, pH 7.5, and target protein was eluted in 6 column volumes 20 mMsodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3.0. Proteinsolution was neutralized by adding 1/10 of 0.5 M sodium phosphate, pH8.0. For in-process analytics after Protein A chromatography, the purityand molecular weight of the molecules in the single fractions wereanalyzed by SDS-PAGE in the absence of a reducing agent and stainingwith Coomassie (InstantBlue™, Expedeon). The NuPAGE® Pre-Cast gel system(4-12% Bis-Tris, Invitrogen) was used according to the manufacturer'sinstruction. Selected fractions of target protein were concentrated andfiltrated prior to loading on a HILOAD® SUPERDEX® 200 column (GEHealthcare) equilibrated with 20 mM histidine, 140 mM sodium chloride,0.01% Tween-20, pH 6.0.

The protein concentration of purified protein samples was determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence.

The aggregate content of the molecules was analyzed using a TSKGEL®G3000 SW XL analytical size-exclusion column (Tosoh) in 25 mM K₂HPO₄,125 mM NaCl, 200 mM L-arginine monohydrocloride, 0.02% (w/v) NaN₃, pH6.7 running buffer at 25° C.

Purity and molecular weight of molecules after the final purificationstep were analyzed by CE-SDS analyses in the presence and absence of areducing agent. The Caliper LABCHIP® GXII system (Caliper lifescience)was used according to the manufacturer's instruction (FIG. 5 and Table3).

Mass spectrometry analysis of the molecules was performed on an AgilentLC-MS system (Agilent Technologies, Santa Clara, Calif., USA). Thechromatography system (Agilent 1260 Infinity) was coupled on an Agilent6224 TOF LC/MS ESI device. About 5 μg of sample were injected on aNUCLEOGEL® RP1000-8, 250 mm×4.6 mm column (MACHEREY-NAGEL GmbH & Co. KG,Duren, Germany) at a flow rate of 1 ml/min at 40° C. The mobile phasewas as follows A: 5% acetonitrile, 0.05% formic acid, and B: 95%acetonitrile, 0.05% formic acid. To apply an elution gradient, 15% B wasraised to 60% B within 10 min, then to 100% B in 2.5 min.

The mass spectrometer was measuring in high resolution mode 4 GHzpositive, and recorded a range from 500 to 3200 m/z. The m/z spectrawere deconvoluted manually with the MassAnalyzer 2.4.1 from Roche(Hoffman-La Roche, Ltd).

All molecules were produced and purified essentially following the samemethod. The final quality was very good for both molecules A and B withalmost 100% monomer content and 100% purity on CE-SDS (Table 2 and 3,FIG. 4). LC-MS analysis was performed for molecule B and revealed nomispairing of light chains. In contrast, molecule F (without chargemodifications) had a very low recovery because side products had to beremoved and LC-MS measurements still showed around 10% mispairing.Molecule C could be purified with a slightly better yield and qualitythan molecule D. The final quality was also good for both molecules Cand D with almost 100% monomer content and >96% purity on CE-SDS (Table2 and 3, FIG. 4).

TABLE 2 Summary of production and purification of anti-CEA/anti-CD3 TCBmolecules with and without charge modifications. Analytical SEC (HMW/Titer Recovery Yield Monomer/LMW) Molecule [mg/l] [%] [mg/l] [%] A 10 404 0/100/0 B 1.5 61 0.9 0.8/99.2/0 C 20 36 7.3 0/100/0 D 10 17 1.7 0/99/1E 16 27 4.3 3/97/0 F 15 3 0.46 0/100/0

TABLE 3 CE-SDS analyses (non-reduced) of anti-CEA/anti-CD3 TCB moleculeswith and without charge modifications. Molecule Peak # Size [kDa] Purity[%] A 1 218.6 100 B 1 199.6 100 C 1 169 100 D 1 98 4 2 166 96 E 1 190 22 200 94 3 210 4 F 1 172 2 2 197 2 3 220 2 4 230 94

Example 3 Comparison of Different Anti-CEA/Anti-CD3 T Cell BispecificMolecule Formats Example 3A Binding of Different Anti-CEA/Anti-CD3 TCell Bispecific (CEA CD3 TCB) Molecules to Cells

The binding of different formats of anti-CEA/anti-CD3 T cell bispecific(CEA CD3 TCB) molecules was tested on human gastric adenocarcinoma cells(MKN45, DSMZ ACC 409, ˜513 000 CEA binding sites), colon adenocarcinomacells (LS174T, ATCC® CL-188, ˜40 700 CEA binding sites), and colonadenocarcinoma cells HT29 (DSMZ ACC 299, ˜10 000 CEA binding sites), aswell as on CD3-expressing immortalized T lymphocyte cells (Jurkat, DSMZACC 282).

Briefly, cells were harvested, counted, checked for viability andresuspended at 2×10⁶ cells/ml in FACS buffer (100 μl PBS 0.1% BSA). 100μl of cell suspension (containing 0.2×10⁶ cells) were incubated inround-bottom 96-well plate for 30 min at 4° C. with increasingconcentrations of the CEA CD3 TCB molecules (310 pM-500 nM), washedtwice with cold PBS 0.1% BSA, re-incubated for further 30 min at 4° C.with the Fluorescein (FITC)-AffiniPure F(ab′)2 Fragment Goat Anti-HumanIgG, Fcγ Fragment Specific antibody (Jackson Immuno Research Lab#109-096-008), washed twice with cold PBS 0.1% BSA.

Staining was fixed by incubation of cells with FACS buffer, containing2% PFA for 30 min at 4° C. in the dark. For the measurement, cells werere-suspended in 150 FACS buffer and fluorescence was measured usingMiltenyi MACSQUANT®

Results are shown in FIG. 5. Binding curves were obtained usingGraphPadPrism5 (upper row from left to right, MKN45 cells; LS174T cells,binding to HT29 cells, lower row, binding to Jurkat cells).

FIG. 5 shows higher fluorescence intensities for molecule D and moleculeC at high concentration on cell lines with medium (LS174T) or low CEAexpression level (HT29). This points to the fact, that more of themonovalently binding constructs are able to bind under these conditionscompared to molecule B, that binds to human CEA bivalently. The bindingto human CD3 on Jurkat cells is comparable for molecule C and moleculeB, whereas molecule D shows better binding. This might be due to betteraccessibility of the CD3-targeting moiety in molecule D.

Example 3B Tumor Cell Killing Induced by Different Anti-CEA/Anti-CD3 TCell Bispecific (CEA CD3 TCB) Molecules

T-cell mediated killing of different tumor cells by CEA CD3 TCBmolecules was assessed using MKN45, BxPC-3 (ECACC 93120816, a humanprimary pancreatic adenocarcinoma cell line) or HT-29 human tumor cellsas targets, and human PBMCs as effector cells. Lysis of tumor cells wasdetected at 24 h and 48 h of incubation with the indicated CEA CD3 TCBmolecules.

Briefly, target cells were harvested with Trypsin/EDTA, washed, andplated at density of 25 000 cells/well using flat-bottom 96-well plates.Cells were left to adhere overnight. Peripheral blood mononuclear cells(PBMCs) were prepared by Histopaque density centrifugation of enrichedlymphocyte preparations (buffy coats) obtained from healthy humandonors. Fresh blood was diluted with sterile PBS and layered overHistopaque gradient (Sigma, # H8889). After centrifugation (450×g, 30minutes, room temperature), the plasma above the PBMC-containinginterphase was discarded and PBMCs transferred in a new falcon tubesubsequently filled with 50 ml of PBS. The mixture was centrifuged(400×g, 10 minutes, room temperature), the supernatant discarded and thePBMC pellet washed twice with sterile PBS (centrifugation steps 350×g,10 minutes). The resulting PBMC population was counted automatically(ViCell) and stored in RPMI1640 medium containing 10% FCS and 1%L-alanyl-L-glutamine (Biochrom, K0302) at 37° C., 5% CO₂ in cellincubator until further use (no longer than 24 h). For the tumor celllysis assay, the CEA CD3 TCB molecules were added at the indicatedconcentrations (range of 1 pM-20 nM in triplicates). PBMCs were added totarget cells at final E:T ratio of 10:1. Target cell killing wasassessed after 24 h and 48 h of incubation at 37° C., 5% CO₂ byquantification of LDH released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100. Minimal lysis (=0%)refers to target cells co-incubated with effector cells withoutbispecific construct.

FIG. 6 shows that molecule D induced the strongest killing of targetcell lines, followed by molecule C and finally molecule B. The bestdifferentiation between the three different formats may be seen on tumorcells lines with low CEA expression levels (FIGS. 6C and 6F). EC50values of tumor cell lysis were calculated using Graph Pad Prism5 andare given in Table 4 (24 h) and Table 5 (48 h).

TABLE 4 EC50 values (pM) for T-cell mediated killing of CEA-expressingtumor cells induced by different CEA CD3 TCB molecules after 24 h. CEAbinding Cell line sites Molecule B Molecule C Molecule D MKN45 513 00078.46 72.96 13.58 BxPC-3  44 400 113.5 91.33 22.19 HT-29  10 000 214.5142.0

TABLE 5 EC50 values (pM) for T-cell mediated killing of CEA-expressingtumor cells induced by different CEA CD3 TCB molecules after 48 h. CEAbinding Cell line sites Molecule B Molecule C Molecule D MKN45 513 00041.24 47.64 11.60 BxPC-3  44 400 46.98 32.42 13.67 HT-29  10 000 31.7263.80 62.44

Example 3C CD25 Up-Regulation on CD4+ and CD8+ Effector Cells afterKilling of CEA-Expressing Tumor Cells Induced by Different CEA CD3 TCBMolecules

Activation of CD4+ (FIG. 7 A-D) and CD8+ T cells (FIG. 7 E-H) afterkilling of CEA-expressing MKN45, BxPC3 or HT29 tumor cells mediated bydifferent CEA CD3 TCB molecules was assessed by FACS analysis usingantibodies recognizing the T cell activation marker CD25 (lateactivation marker). In addition, CD25 was analyzed on CD4 and CD8 Teffector cells upon co-incubation of effector cells with the differentCEA CD3 TCB molecules in the absence of target cells to check forantigen-unspecific T cell activation.

The antibody and the killing assay conditions were essentially asdescribed above (Example 3B), using the same antibody concentrationrange (1 pM-20 nM in triplicates), E:T ratio 10:1 and an incubation timeof 48 h.

After the incubation, PBMCs were transferred to a round-bottom 96-wellplate, centrifuged at 350×g for 5 min and washed twice with PBScontaining 0.1% BSA. Surface staining for CD8 (FITC anti-human CD8,BioLegend #344704), CD4 (PECy7 anti-human CD4, BioLegend #344612) andCD25 (APC anti-human CD25 BioLegend #302610) was performed according tothe suppliers' indications. Cells were washed twice with 150 μl/well PBScontaining 0.1% BSA and fixed for 30 min at 4° C. using 150 μl/well ofFACS buffer, containing 2% PFA. After centrifugation, the samples werere-suspended in 200 μl/well PBS 0.1% BSA and analyzed using a BD FACSFortessa.

FIG. 7 shows that molecule D induced the strongest T cell activation, asmeasured by percentage of CD25-positive T cells. However, this moleculealso induced activation of T cells in the absence of target cells at thehighest 2-3 concentrations and was therefore not selected as preferredformat. Molecule C is the second most potent molecule to induce T cellactivation in the presence of CEA-expressing target cells, which isespecially apparent in settings with target cells that express ratherlow levels of CEA (FIG. 7C). Also molecule B is able to induce strongand concentration-dependent T cell activation in the presence ofCEA-expressing target cells. In this example, both molecules B and C donot induce significant T cell activation in the absence of target cells.

To further elaborate this important safety point, additional assays wereperformed.

Example 3D CD69 Up-Regulation on CD4+ and CD8+ Effector Cells UponCo-Incubation with Different CEA

CD3 TCB molecules and CEA-expressing tumor or primary epithelial cellsActivation of CD4+ (FIG. 8 A-E) and CD8+ T cells (FIG. 8 F-J) wasassessed after co-incubation of CEA-expressing MKN45, LS174T (ECACC87060401, a human colon adenocarcinoma cell line with approximately 40700 CEA binding sites), HT29 or CCD 841 CoN (ATCC® CRL-1790™, a primaryepithelial cell line from human colon, expressing <2000 CEA bindingsites) with human PBMCs and different CEA CD3 TCB molecules for 48 h. Tocheck for antigen-unspecific T cell activation, CD69 was analyzed on CD4and CD8 T effector cells upon co-incubation of effector cells with thedifferent CEA CD3 TCB molecules in the absence of target cells as well.

The antibody and assay conditions were essentially as described above(Example 3B and 3C), using the same antibody concentration range (1pM-20 nM in triplicates) and an E:T ratio of 10:1, as well as the FACSstaining protocol as described above (PE anti-human CD69, BioLegend#310906). FIG. 8 shows that again molecule D induced the strongest Tcell activation, as measured by percentage of CD69-positive T cells inthe presence of different CEA-expressing tumor cells. However, thismolecule also induced activation of T cells in the presence of primaryepithelial cells (CCD841, see FIGS. 8D and 8I), as well as in theabsence of target cells (FIGS. 8E and 8J). These findings confirm theantigen-independent activation of T cells and in addition point to apotential safety issue in the presence of primary epithelial cells withvery low CEA expression levels.

However, with these reactive PBMC effector cells, antigen-independent Tcell activation was observed with the second most potent molecule aswell, molecule C. Consequently, the only format that showed strong andconcentration-dependent killing of various CEA-expressing tumor cells,but no significant killing of primary epithelial cells, norantigen-independent T cell activation in the presence of primaryepithelial cells or in the absence of target cells is molecule B.Therefore, the “2+1 IgG CrossFab, inverted” format was selected as thepreferred format.

Example 4 Comparison of Anti-CEA/Anti-CD3 T Cell Bispecific MoleculesComprising Parental or Humanized CEA Binders Example 4A T CellActivation and Tumor Cell Killing Induced by Different CEA CD3 TCBMolecules (Chimeric T84.66 Versus Humanized Variant 1)

The impact of the CEA binder (parental chimeric T84.66 versus humanizedvariant 1) on the final potency of the CEA CD3 TCB to induce T cellactivation or tumor cell lysis was assessed with a classical tumor celllysis assay with subsequent staining of T cell activation markers asdescribed above (Example 3B and Example 3C). To further evaluate thesafety window, both molecules were co-incubated with effector cells onlyas well.

Briefly, target cells included in this assay were MKN45 and CCD841 CoNcells. The CEA CD3 TCB molecules A and B were added at the indicatedconcentration range of 1 pM-20 nM (in triplicates). PBMCs were added totarget cells at a final E:T ratio of 10:1. Lysis of tumor or primaryepithelial cells was detected at 48 h of incubation with the indicatedCEA CD3 TCB molecules. PBMCs of this assay were harvested and stainedfor CD8+ and the early activation marker CD69 as described above(Example 3C).

FIG. 9 shows that the CEA CD3 TCB molecule containing the parentalchimeric T84.66 CEA binder (molecule A) induced both T cell activation(FIG. 9 A, B) and cell lysis (FIG. 9 C, D) not only in the presence ofCEA high expressing tumor cells (MKN45), but also in the presence ofprimary epithelial cells CCD841 with very low CEA expression levels orin the absence of target cells.

In contrast, the CEA CD3 TCB molecule containing the humanized binder(molecule B) induced T cell activation only in the presence of the tumorcell line MKN45, but not in the presence of the primary epithelial cellline. The same is true for cell lysis. In addition, there was no sign ofsignificant T cell activation in the absence of target cells.

Example 4B

To further evaluate the safety window of the CEA CD3 TCB molecules,containing either the parental, chimeric T84.66 CEA binder (molecule A)or the humanized variant 1 (molecule B), a sensitive Jurkat-NFATreporter assay was conducted. In principle, the simultaneous binding ofthe TCB molecule to human CEA on antigen-expressing cells and to humanCD3 on Jurkat-NFAT reporter cells (a human acute lymphatic leukemiareporter cell line with a NFAT promoter-regulated luciferase expression,GloResponse Jurkat NFAT-RE-luc2P, Promega # CS176501) the NFAT promoteris activated and leads to expression of active firefly luciferase. Theintensity of the luminescence signal (obtained upon addition ofluciferase substrate) is proportional to the intensity of CD3 activationand signaling.

For the assay, target cells were harvested with trypsin/EDTA andviability was determined using ViCell. 20 000 cells/well were plated ina flat-bottom, white-walled 96-well-plate (#655098, greiner bio-one) anddiluted antibodies or medium (for controls) was added at the indicatedconcentration range (0.4 pM-100 nM). Subsequently, Jurkat-NFAT reportercells were harvested and viability assessed using ViCell. Cells wereresuspended in cell culture medium and added to tumor cells to obtain afinal E:T of 2.5:1 and a final volume of 100 μl per well. Cells wereincubated for 4 h at 37° C. in a humidified incubator. At the end of theincubation time, 100 μl/well of ONE-Glo solution (1:1 ONE-Glo and assaymedium volume per well) were added to wells and incubated for 10 min atroom temperature in the dark. Luminescence was detected using WALLACVictor3 ELISA reader (PerkinElmer2030), 1 sec/well as detection time.

FIG. 10 shows that the CEA CD3 TCB molecule containing the parentalchimeric T84.66 CEA binder (molecule A) induced Jurkat T cell activationnot only in the presence of CEA high and medium expressing tumor cells(MKN45 and LS174T respectively), but also in the presence of primaryepithelial cells CCD841 with very low CEA expression levels or in theabsence of target cells (FIG. 10A).

In contrast, the CEA CD3 TCB molecule containing the humanized binder(molecule B) induced Jurkat T cell activation only in the presence ofthe tumor cell lines MKN45 and LS174T, but not in the presence of theprimary epithelial cell line or in the absence of targets (FIG. 10B).These results surprisingly show that the molecule comprising thehumanized CEA binder is better in terms of safety.

Example 5 Comparison of Anti-CEA/Anti-CD3 T Cell Bispecific MoleculesComprising Different Humanized CEA Binders Example 5A T Cell Activationand Tumor Cell Killing Induced by CEA CD3 TCB Molecules ComprisingDifferent Humanized CEA Binders

The impact of the CEA binder (humanized variant 1 (SEQ ID NOs 22 and 23)versus a different humanized CEA binder (SEQ ID NOs 30 and 31, not basedon T84.66)) on the final potency of the CEA TCB to induce tumor celllysis was assessed with a classical tumor cell lysis assay as describedabove (Example 3B).

Briefly, target cells included in this assay were BxPC-3, NCI-H2122(ATCC® CRL-5985, a human non-small cell lung cancer cell line, ˜13 300CEA binding sites), COR-L105 (Sigma-Aldrich #92031918, a human lungadenocarcinoma cell line, ˜1200 CEA binding sites) and HBEpiC (ChemieBrunschwig AG #3210, human bronchial epithelial cells, <500 CEA bindingsites). The CEA CD3 TCB molecule comprising the humanized variant 1 CEAbinder (molecule B) was added at the indicated concentration range of 1pM-20 nM (in triplicates), the CEA CD3 TCB molecule comprising thedifferent humanized CEA binder (i.e. a CEA CD3 TCB molecule of similarstructure as molecule B, but comprising a different CEA binder, see SEQID NOs 42-45) was added at the indicated concentration range of 6 pM-100nM. PBMCs were added to target cells at a final E:T ratio of 10:1. Lysisof tumor or primary epithelial cells was detected at 47 h of incubationwith the indicated CEA CD3 TCB molecules.

Subsequently, PBMCs of this assay were harvested and stained for theearly activation marker CD69 on human CD8+ T cells as described above.

FIG. 11 A-D shows that the CEA CD3 TCB based on humanized variant 1(molecule B of Example 2) induced stronger T cell activation uponsimultaneous binding to T effector and CEA-positive target cellscompared to the TCB molecule based on the different humanized CEA binder(referred to in the following as “molecule X”, SEQ ID NOs 42-45).

This is in line with the tumor cell lysis data depicted in FIG. 11 E-H,where stronger killing of CEA-expressing tumor cells was observed withthe molecule based on humanized variant 1.

Remarkably, none of the CEA CD3 TCB molecules induced lysis of CEA-lowprimary epithelial HBEpiC cells.

Taken together, the CEA CD3 TCB molecule based on humanized variant 1(molecule B) is able to kill tumor cells with much lower CEA levels ascompared to the CEA CD3 TCB based on a different humanized CEA binder(molecule X), while maintaining the safety window.

Example 5B T Cell Proliferation Induced by CEA CD3 TCB MoleculesComprising Different Humanized CEA Binders

As an alternative read-out, the TCB molecules used in Example 5A wereanalyzed for their capability to induce T cell proliferation uponcross-linkage in the presence of the respective tumor target cells(MKN45, LS174T, HT29). As a control, primary epithelial cells CCD841 CoNwith very low CEA expression levels were included as alternative targetcells as well.

Briefly, freshly isolated human PBMCs were adjusted to 1 million cellsper ml in warm PBS and stained with 0.1 μM CFSE in a humidifiedincubator at 37° C. for 15 minutes. The staining was stopped by additionof 1/10 volume of FCS, that was incubated for 1 min at room temperature.Subsequently, the cells were centrifuged, re-suspended in pre-warmedmedium and incubated for another 30 min in a humidified incubator at 37°C. to remove remaining CFSE. After the incubation the cells were washedonce with warm medium, counted and re-suspended in medium at 2 mio cellsper ml.

0.02 million target cells were plated per well of a flat-bottom 96-wellplate and the different TCB molecules were added at the indicatedconcentrations. CFSE-labeled PBMCs were added to obtain a final E:Tratio of 10:1 and the assay plates were incubated for five days in ahumidified incubator at 37° C.

On day five, the effector cells were harvested, washed twice with FACSbuffer (PBS, 0.1% BSA) and stained for surface expression of CD4 andCD8. Proliferation of the different T cell subpopulations was analyzedusing a BD FACS Fortessa, equipped with PD FACS Diva Software.Proliferation curves were analyzed by GraphPadPrism5.

FIG. 12 shows that the CEA CD3 TCB based on humanized variant 1(molecule B) induced stronger T cell activation and subsequent T cellproliferation in the presence of CEA-positive tumor target cellscompared to the CEA CD3 TCB based on the different humanized CEA binder(molecule X). Proliferation of CD8+ T cells is shown in FIG. 12 A-D,proliferation of CD4+ T cells in FIG. 12 E-H.

Notably, even after 5 days of incubation no sign of significant T cellactivation and subsequent T cell proliferation could be observed witheither one of the molecules in the presence of primary epithelial cells(FIG. 12, CCD841 CoN cells).

This further confirms the favorable potency and safety window of the CEACD3 TCB based on humanized variant 1 (molecule B), even as compared tothe CEA CD3 TCB based on a different humanized CEA binder (molecule X).

Example 5C Binding of Different Anti-CEA/Anti-CD3 T Cell Bispecific (CEACD3 TCB) Molecules to Cells

The binding of the TCB molecules used in Example 5A and B was tested ondifferent CEA-expressing tumor and CD3-expressing Jurkat (DSMZ ACC 282)cells.

Briefly, cells were harvested, counted, checked for viability andre-suspended at 2×10⁶ cells/ml in FACS buffer (100 μl PBS 0.1% BSA). 100μl of cell suspension (containing 0.2×10⁶ cells) were incubated inround-bottom 96-well plate for 30 min at 4° C. with increasingconcentrations of the CEA IgG (31 pM-500 nM), washed twice with cold PBS0.1% BSA, re-incubated for further 30 min at 4° C. with theFITC-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgG FcgFragment Specific secondary antibody (Jackson Immuno Research Lab FITC#109-096-008, 1:40 pre-diluted in PBS 0.1% BSA), washed twice with coldPBS 0.1% BSA and fixed using 150 μl PBS 0.1% BSA, containing 2% PFA andincubation at 4° C. for 20 min. Thereafter, cells were washed once for 8min at 400×g, 4° C. and finally re-suspended in 150 μl FACS buffer forthe FACS measurement. Fluorescence was measured using MiltenyiMACSQuant.

Binding curves and EC50 values were obtained and calculated usingGraphPadPrism6 (FIG. 16 A, binding to MKN45 cells, FIG. 16 B, binding toLS-174T cells, FIG. 16 C, binding to HT-29 cells, FIG. 16 D, Table 6).

FIG. 16 shows, that the CEA CD3 molecule based on humanized variant 1(molecule B) displays better binding to CEA-expressing tumor cells thanthe CEA CD3 TCB based on a different humanized CEA binder (molecule X)(better EC50 values and maximal binding, particularly on medium and lowCEA-expressing target cells). Both TCB molecules showconcentration-dependent binding to human CD3 on Jurkat cells.

TABLE 6 Binding of CEA CD3 TCB molecule B and CEA CD3 TCB molecule X tocells (EC50 values, based on binding curves shown in FIG. 16, calculatedby Graph Pad Prism). CEA Molecule B Molecule X binding EC50 binding EC50binding Cell Line Vendor sites (nM) (nM) MKN45 DSMZ ~513 300 26.61 27.85#ACC409 LS-174T ATCC ®  ~40 700 4.256 11.69 #CL-188 HT-29 DSMZ  ~10 00018.66 n.c. #ACC299

CEA binding sites were determined by a FACS-based Qifikit analysis,according to the manufacturers' instructions, using 10 μg/ml anti-humanCEA antibody (Santa Cruz Biotechnology, sc-23928).

Example 5D Tumor Cell Lysis Induced by CEA CD3 TCB Molecules ComprisingDifferent Humanized CEA Binders

T-cell mediated lysis of different tumor cells by the CEA CD3 TCBmolecules used in Examples 5A-C was assessed using human tumor cells astarget cells, and human PBMCs as effector cells. Lysis of tumor cellswas detected at 48 h of incubation with the indicated CEA CD3 TCBmolecules. Briefly, target cells were harvested with Trypsin/EDTA,washed, and plated at density of 25 000-000 cells/well using flat-bottom96-well plates. Cells were left to adhere overnight. Peripheral bloodmononuclear cells (PBMCs) were prepared by Histopaque densitycentrifugation of enriched lymphocyte preparations (buffy coats)obtained from healthy human donors. Fresh blood was diluted with sterilePBS and layered over Histopaque gradient (Sigma, # H8889). Aftercentrifugation (450×g, 30 minutes, room temperature), the plasma abovethe PBMC-containing interphase was discarded and PBMCs transferred in anew falcon tube subsequently filled with 50 ml of PBS. The mixture wascentrifuged (400×g, 10 minutes, room temperature), the supernatantdiscarded and the PBMC pellet washed twice with sterile PBS(centrifugation steps 350×g, 10 minutes). The resulting PBMC populationwas counted automatically (ViCell) and stored in RPMI1640 mediumcontaining 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37°C., 5% CO₂ in a cell incubator until further use (no longer than 24 h).For the tumor cell lysis assay, the CEA CD3 TCB molecules were added atthe indicated concentrations (range of 0.26 pM-20 nM for CEA CD3 TCBbased on humanized variant 1 (molecule B), respective 1.28 pM-100 nM forCEA CD3 TCB based on different humanized CEA binder (molecule X), intriplicates). PBMCs were added to target cells at final E:T ratio of10:1. Target cell killing was assessed after 48 h of incubation at 37°C., 5% CO₂ by quantification of LDH released into cell supernatants byapoptotic/necrotic cells (LDH detection kit, Roche Applied Science, #11644 793 001). Maximal lysis of the target cells (=100%) was achieved byincubation of target cells with 1% Triton X-100. Minimal lysis (=0%)refers to target cells co-incubated with effector cells withoutbispecific construct.

FIG. 17 shows that the CEA CD3 TCB molecule based on humanized variant 1(molecule B) induced significant and concentration-dependent lysis ofall shown tumor cell lines, whereas the CEA CD3 TCB based on thedifferent humanized CEA binder (molecule X) induced tumor lysis ofKatoTI and to a lesser extent of NCT-H2122 only. This clearlydemonstrates the higher potency of molecule B as compared to molecule X,especially for tumor cell lines with rather low CEA expression levels.

EC50 values of tumor cell lysis were calculated using Graph Pad Prism6and are given in Table 7 (48 h).

TABLE 7 EC50 values (pM) for T-cell mediated lysis of low CEA-expressingtumor cells induced by different CEA CD3 TCB molecules after 48 h. CEAMolecule Molecule Tumor binding B EC50 X EC50 Cell Line IndicationReference sites lysis lysis Kato III Gastric ECACC ~22 700 55.4 8233Cancer #86093004 HCC1954 Breast ATC ~15 750 421.7 — Cancer #CRL-2338NCI-H2122 Lung ATCC ~13 300 27.9 — Cancer #CRL-598 CX-1 Colon DSMZ  ~4750 430.4 — Cancer #ACC 129 NCI-H596 Lung ATCC  ~1 900 18.5 — Cancer#HTB-178

CEA binding sites were determined by a FACS-based Qifikit analysis,according to the manufacturers' instructions, using 10 μg/ml anti-humanCEA antibody (Santa Cruz Biotechnology, sc-23928).

Example 6 Comparison of Anti-CEA/Anti-CD3 T Cell Bispecific MoleculesComprising Different Linkers Between CEA and CD3 Binders Example 6ABinding of CEA CD3 TCB Molecules Comprising Different Linkers BetweenCEA and CD3 Binders to Cells

The binding of a variant of molecule B with a longer linker between theCD3 Fab and the CEA Fab (molecule E) was compared to molecule B for itsbinding to cells.

The binding to human CEA was tested on MKN45, LS174T or HT29, thebinding to human CD3 was tested on Jurkat cells. The assay set-up andconditions were as described above (Example 3A).

Results are shown in FIG. 13. Binding curves were obtained usingGraphPadPrism5 (A, binding to MKN45 cells; B, binding to LS174T cells,C, binding to HT29 cells, D, binding to Jurkat cells).

FIG. 13 shows comparable binding of both molecules to human CEA, as wellas to human CD3 on cells.

Example 6B Lysis of Various Tumor Cells by CEA CD3 TCB MoleculesComprising Different Linkers Between CEA and CD3 Binders

To further evaluate the impact of the linker length on the potency ofthe molecule to induce T cell-mediated lysis of CEA-expressing tumorcells, a classical tumor cell lysis assay was performed, as describedabove (e.g. in Example 3B). The CEA CD3 TCB molecules of Example 6A(molecule B and a corresponding molecule with a longer linker betweenthe CEA and CD3 binders, molecule E) were added at the indicatedconcentrations (range of 1 pM-20 nM in triplicates) and tumor cell lysiswas assessed after 24 h (FIG. 14 A-D) and 48 h (FIG. 14 E-H). EC50values were calculated by GraphPadPrism5 and are given in Table 8.

FIG. 14 shows comparable lysis of tumor cells expressing high (MKN45) ormedium levels of CEA (LS174T) and no killing of primary epithelial cellsCCD841.

However, target cells with low CEA expression levels (HT29) showedhigher overall killing with the molecule comprising the longer linker.

TABLE 8 EC50 values (pM) for T-cell mediated killing of lowCEA-expressing HT29 tumor cells induced by different CEA CD3 TCBmolecules after 24 h and 48 h. EC50 (pM) Molecule B Molecule E 24 h 512679 48 h 338 342

Example 7 Tumor Cell Lysis and T Cell Activation Induced by CEA CD3 TCBMolecule Example 7A

Lysis of Cell Lines with Different CEA Expression Levels by CEA CD3 TCBMolecule B

In another experiment (FIG. 18A), the CEA CD3 TCB molecule B wascharacterized in the presence of the low CEA-expressing primaryepithelial cell line HBEpiC versus different tumor cell lines to assessits safety. The assay set-up and antibody range was as described inExample 5D for molecule B. EC50 values of tumor cell lysis werecalculated using Graph Pad Prism6 and are given in Table 9 (48 h).

As depicted in FIG. 18A and Table 9, primary epithelial cells were notkilled by the CEA CD3 TCB molecule, whereas tumor cell lines withvarying CEA expression levels could be lysed by the CEA CD3 TCB moleculein a concentration-dependent manner.

TABLE 9 EC50 values (pM) for T-cell mediated lysis of CEA-expressingtumor cells induced by CEA CD3 TCB molecule B after 48 h. CEA bindingCell Line Reference sites Molecule B BxPC-3 ECACC ~44 000 49.63#93120816 NCI-H2122 ATCC ~13 300 237.5 #CRL-598 COR-L105 Sigma-Aldrich ~1 200 n.c.* #92031918 HBEpiC ScienCell #3210   <600 — *The EC50 forCOR-L105 could not be calculated properly because the curve did notreach saturation at high concentrations.

CEA binding sites were determined by a FACS-based Qifikit analysis,according to the manufacturers' instructions, using 10 μg/ml anti-humanCEA antibody (Santa Cruz Biotechnology, sc-23928.

In another experiment (FIG. 19A), the CEA CD3 TCB molecule B wascharacterized in the presence of another low CEA-expressing primaryepithelial cell line (CCD841CoN) versus different tumor cell lines toassess its safety, using another PBMC donor. The assay set-up andantibody range was as described in Example 5D for the CEA CD3 TCBmolecule B. EC50 values of tumor cell lysis were calculated using GraphPad Prism6 and are given in Table 10 (48 h).

TABLE 10 EC50 values (pM) for T-cell mediated lysis of CEA-expressingtumor cells induced by CEA CD3 TCB molecule B after 48 h. CEA bindingCell Line Reference sites Molecule B MKN45 DSMZ ~513 300 41.24 #ACC409BxPC-3 ECACC  ~44 000 46.98 #93120816 HT-29 DSMZ  ~10 000 562.7 #ACC299CCD- ATCC    <600 — 841CoN #CRL-1790

CEA binding sites were determined by a FACS-based Qifikit analysis,according to the manufacturers' instructions, using 10 μg/ml anti-humanCEA antibody (Santa Cruz Biotechnology, sc-23928).

As depicted in FIG. 19A and Table 10, primary epithelial cells were notkilled by CEA CD3 TCB molecule B, whereas tumor cell lines with varyingCEA expression levels could be lysed by the CEA CD3 TCB molecule in aconcentration-dependent manner.

Example 7B

CD69 Up-Regulation on CD4+ Effector Cells after Killing ofCEA-Expressing Tumor Cells Induced by CEA CD3 TCB Molecule B

Activation of CD4+ and CD8+ effector cells after lysis of CEA-expressingtumor cells mediated by the CEA CD3 TCB molecule B was assessed by FACSanalysis using antibodies recognizing the T cell activation marker CD69(early activation marker).

The antibody and the killing assay conditions were essentially asdescribed above (Example 5D), using the same antibody concentrationrange (0.26 pM-20 nM in triplicates), E:T ratio 10:1 and an incubationtime of 48 h.

After the incubation, PBMCs were transferred to a round-bottom 96-wellplate, centrifuged at 350×g for 5 min and washed twice with PBScontaining 0.1% BSA. Surface staining for CD8 (FITC anti-human CD8, BDBiosciences #555634), CD4 (PECy7 anti-human CD4, BD Biosciences #557852)and CD69 (PE anti-human CD69 BioLegend #310906) was performed accordingto the suppliers' indications. Cells were washed twice with 150 μl/wellPBS containing 0.1% BSA and fixed for 30 min at 4° C. using 150 μl/wellof FACS buffer, containing 2% PFA. After centrifugation, the sampleswere re-suspended in 200 μl/well PBS 0.1% BSA and analyzed using a BDFACS Fortessa.

FIG. 18B shows that the CEA CD3 TCB molecule B inducedconcentration-dependent T cell activation in the presence of differentCEA-expressing tumor cell lines, as measured by percentage ofCD69-positive CD4+ T cells. In contrast, no T cell activation occurredin the presence of low CEA-expressing primary epithelial cells. Similarresults were obtained for CD8+ cells (data not shown). The data suggeststhat therapeutic administration of CEA CD3 TCB molecule B should notlead to adverse effects on primary epithelial cells with low CEAexpression levels.

EC50 values of T-cell activation were calculated using Graph Pad Prism6and are given in Table 11.

TABLE 11 EC50 values (pM) for T-cell activation upon simultaneousbinding of CEA CD3 TCB molecule B to CEA-expressing cells andCD3-expressing T cells after 48 h CEA binding Cell Line Reference sitesMolecule B BxPC-3 ECACC ~44 000 84.74 #93120816 NCI-H2122 ATCC ~13 300149.1 #CRL-598 COR-L105 Sigma-Aldrich  ~1 200 n.c.* #92031918 HBEpiCScienCell #3210   <600 — *The EC50 for COR-L105 could not be calculatedproperly because the curve did not reach saturation at highconcentrations.

CEA binding sites were determined by a FACS-based Qifikit analysis,according to the manufacturers' instructions, using 10 μg/ml anti-humanCEA antibody (Santa Cruz Biotechnology, sc-23928).

Example 7C

CD25 Up-Regulation on CD4+ Effector Cells after Killing ofCEA-Expressing Tumor Cells Induced by CEA CD3 TCB Molecule B

Activation of CD4+ and CD8+ after lysis of CEA-expressing tumor cellsmediated by the CEA CD3 TCB molecule B was assessed by FACS analysisusing antibodies recognizing the T cell activation marker CD25 (lateactivation marker).

The antibody and the killing assay conditions were essentially asdescribed above (Example 5D), using the same antibody concentrationrange (0.26 pM-20 nM in triplicates), E:T ratio 10:1 and an incubationtime of 48 h.

After the incubation, PBMCs were transferred to a round-bottom 96-wellplate, centrifuged at 350×15 g for 5 min and washed twice with PBScontaining 0.1% BSA. Surface staining for CD8 (FITC anti-human CD8,BioLegend #344704), CD4 (PECy7 anti-human CD4, BioLegend #344612) andCD25 (APC anti-human CD25 BioLegend #302610) was performed according tothe suppliers' indications. Cells were washed twice with 150 μl/well PBScontaining 0.1% BSA and fixed for 30 min at 4° C. using 150 μl/well ofFACS buffer, containing 2% PFA. After centrifugation, the samples werere-suspended in 200 μl/well PBS 0.1% BSA and analyzed using a BD FACSFortessa.

FIG. 19B shows that the CEA CD3 TCB molecule B inducedconcentration-dependent T cell activation in the presence ofCEA-expressing tumor cells, as measured by percentage of CD25-positive Tcells. In contrast, no T cell activation occurred in the presence of lowCEA-expressing primary epithelial cells. Similar results were obtainedfor CD8+ cells (data not shown). The data suggests that therapeuticadministration of CEA CD3 TCB molecule B should not lead to adverseeffects on primary epithelial cells with low CEA expression levels.

Example 8 Specific Binding of CEA CD3 Molecule B to Human CEACAM5

To show the specific binding of the CEA CD3 TCB molecule B to humanCEACAM5, but not to the other closest family members CEACAM1 andCEACAM6, binding of CEA CD3 TCB molecule B to transient HEK293Ttransfected cells, expressing either human CEACAM5, CEACAM1 or CEACAM6,was evaluated.

Briefly, cells were harvested, counted, checked for viability andre-suspended at 1×10⁶ cells/ml in FACS buffer (100 μl PBS 0.1% BSA). 100μl of the cell suspension (containing 0.1×10⁶ cells) were plated itonround-bottom 96-well plates and washed twice with 150 μl of cold PBS.Cells were stained for 30 min at 4° C., using a 1:5000 pre-dilutedsuspension of the fixable viability dye eFluor660 (eBioscience,#65-0864-14) in PBS. Thereafter, the cells were washed twice with PBS,once with FACS buffer and stained for 30 min at 4° C. with increasingconcentrations of the CEA CD3 TCB molecule B. The antibody concentrationrange was 30.5 pM-500 nM. Cells were washed twice with cold PBS 0.1%BSA, re-incubated for further 30 min at 4° C. with the FITC-conjugatedAffiniPure F(ab′)2 Fragment goat anti-human IgG Fcg Fragment Specificsecondary antibody (Jackson Immuno Research Lab FITC #109-096-098, 1:50pre-diluted in PBS 0.1% BSA), washed twice with cold PBS 0.1% BSA andfixed using 150 μl PBS 0.1% BSA, containing 2% PFA and incubation at 4°C. for 20 min. Thereafter, cells were washed once for 8 min at 400×g, 4°C. and finally re-suspended in 150 μl FACS buffer for the FACSmeasurement. Fluorescence was measured using BD FACS CantoII. Bindingcurves were obtained using GraphPadPrism6 (FIG. 20A).

To determine the transfection efficacy, all transfectants were stainedfor 30 min at 4° C., using a commercially available anti-human CD66antibody (FITC mouse anti-human CD66, BD Biosciences #551479, 20 μl persample) (FIG. 20B). As shown in FIG. 20B, the highest expression levelwas detected for human CEACAM1, followed by CEACAM5 and CEACAM6.

FIG. 20A clearly shows that the CEA CD3 TCB molecule B showsconcentration-dependent binding to transient transfectants expressinghuman CEACAM5, but not to any of the other transfectants, expressinghuman CEACAM1 or CEACAM6, demonstrating the specificity of the bindingto CEA.

Example 9 Single Dose PK of CEA CD3 TCB Molecules Comprising DifferentHumanized CEA Binders in Healthy NOG Mice

Single dose pharmacokinetic studies (SDPK) were performed in healthy NOGmice to evaluate exposure of CEA CD3 TCB molecule B and CEA CD3 TCBmolecule X (FIG. 15). An intravenous (i.v.) bolus injection of 0.5 mg/kgwas administered to NOG mice and blood samples were taken at selectedtime points for pharmacokinetic evaluation. A generic immunoassay wasused for measuring total concentrations of CEA CD3 TCB molecules. Thecalibration range of the standard curve for both TCB molecules was 0.078to 5 ng/ml, where 1.5 ng/ml is the lower limit of quantification (LLOQ).

A biphasic decline was observed with a half-life of 10 days(non-compartmental analysis) for CEA CD3 TCB molecule B and 6.5 days forCEA CD3 TCB molecule X. A clearance (CL) of 8.1 ml/d/kg was detected forCEA CD3 TCB molecule B and 19 ml/d/kg for CEA CD3 TCB molecule X,respectively (Table 12). Overall, CEA CD3 TCB molecule B showed longerhalf-life and lower CL in NOG mice compared to CEA CD3 TCB molecule X.

The Phoenix v6.4 from Pharsight Ltd was used for PK analysis, modellingand simulation.

TABLE 12 Pharmacokinetic parameters of a 0.5 mg/kg iv bolusadministration of CEA CD3 TCB molecules in NOG mice. Half-life CLConstruct (d) (mL/d/kg) Molecule B 10 8.1 Molecule X 6.5 19

Example 10 Anti-Tumor Activity of CEA CD3 TCB Molecules ComprisingDifferent Humanized CEA Binders in the MKN45 Model

Anti-tumor activity of CEA CD3 TCB molecule B and CEA CD3 TCB molecule Xwas tested in fully humanized NOD/Shi-scid/IL-2Rγ^(null) (NOG) micebearing the gastric carcinoma cell line MKN45.

Fully humanized NOG mice at 14 weeks of age, bearing physiologicallevels of circulating human B- and T-cells (Hayakawa et al., Stem Cells27 (2009) 175-182), were injected sub-cutaneous (s.c.) with 1×10⁶ MKN45cells (originally obtained from the DSMZ-Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH). When average tumor volumereached 100 mm³, mice received CEA CD3 TCB molecule X or CEA CD3 TCBmolecule B i.v. at the dose of 2.5 mg/kg administered either twice oronce a week, and at the dose of 0.5 mg/kg administered once a week (FIG.21). CEA CD3 TCB molecule B showed significantly stronger anti-tumoractivity at all tested doses and schedules. Importantly, only CEA CD3TCB molecule B could mediate tumor regression with tumor-free micedetected in the 2.5 mg/kg and 0.5 mg/kg treated groups (FIG. 21, Table13).

TABLE 13 Number of tumor-free mice per group at study termination (day70). Tumor-free mice at termination Treatment (study day 70) Vehicle 0/9Molecule X 2.5 mg/kg twice/week 0/9 Molecule B 2.5 mg/kg twice/week 3/9Molecule X 2.5 mg/kg once/week 0/9 Molecule B 2.5 mg/kg once/week 1/9Molecule X 0.5 mg/kg once/week 0/9 Molecule B 0.5 mg/kg once/week 1/8

Example 11

Based on the SDPK data shown in Example 9, a 2-compartmental model wascompiled to describe the PK of CEA CD3 TCB molecule B and CEA CD3 TCBmolecule X in NOG mice (FIG. 22). The different dose levels andschedules selected for the dose-range efficacy study described in thenext example were simulated. As shown in FIG. 22, to compensate for thelower clearance of CEA5 CD3 TCB molecule B the design of the study wasadapted. The frequency of administration for CEA CD3 TCB molecule X wasincreased to a twice weekly schedule and also higher dose levels up to12.5 mg/kg were used. The simulated profiles show that at a bi-weeklydose of 12.5 mg of CEA CD3 TCB molecule X the exposure is substantiallyhigher as compared to 0.5 mg/kg CEA CD3 TCB molecule B.

Example 12 Dose-Range Efficacy Study with CEA CD3 TCB MoleculesComprising Different Humanized CEA Binders in the MKN45 Model

To more specifically assess the in vivo fold difference in anti-tumoractivity between the two molecules, CEA CD3 TCB molecule B and CEA CD3TCB molecule X were tested at a larger range of doses in fully humanizedNSG mice bearing the gastric carcinoma cell line MKN45 (FIG. 23).

Fully humanized NSG mice were injected s.c. with 1×10⁶ MKN45 cells. Whenaverage tumor volume reached 180 mm³, mice received CEA CD3 TCB moleculeB or CEA CD3 TCB molecule X i.v. at the different doses and schedulesdepicted in FIG. 23. The doses and schedules were selected based on theanalysis described in Example 11. The efficacy data obtained clearlyshow that CEA CD3 TCB molecule B could mediate stronger anti-tumoractivity when compared to CEA CD3 TCB molecule X with a significant folddifference of at least 25 times (as highlighted by the star in the graphon FIG. 23).

Example 13 Anti-Tumor Activity of CEA CD3 TCB Molecules ComprisingDifferent Humanized CEA Binders in the HPAF-II Model

Anti-tumor activity of CEA CD3 TCB molecule B and CEA CD3 TCB molecule Xwas tested in NOD.Cg-Prkdc^(scid) Il2rg^(tmlWjl)/SzJ (NSG) mice bearingthe human pancreatic carcinoma cell line HPAF-II and transferred withhuman peripheral mononuclear cells (PBMC). Briefly, female NOG mice wereinjected sub-cutaneously (s.c.) with 1×10⁶ HPAF-II cells (originallyobtained from the American Type Culture Collection (ATCC)). When averagetumor volume reached 150 mm³, mice received i.v. injection of human PBMC(10×10⁶ cells per mouse) as source of human T-cells. Three days later,mice received CEA CD3 TCB molecule B or CEA CD3 TCB molecule X i.v. at adose of 2.5 mg/kg, administered once a week. As depicted in FIG. 24,after 3 weeks treatment, both TCB molecules show potent anti-tumoractivity, with only molecule B able to mediate tumor regression (at day32, study day termination) (FIG. 24).

Example 14 Single Dose PK of CEA CD3 TCB Molecules Comprising DifferentHumanized CEA Binders in Cynomolgus Monkeys

Single dose pharmacokinetic studies (SDPK) were performed in cynomolgusmonkeys to assess the exposure of CEA CD3 TCB molecule B and CEA CD3 TCBmolecule B, respectively (FIG. 25). An IV bolus administration of 0.01mg/kg was administered and blood samples were taken at selected timepoints for pharmacokinetic evaluation. Specific immunoassays were usedmeasuring binding competent concentrations of CEA CD3 TCB molecule B andCEA CD3 TCB molecule X. For CEA CD3 TCB molecule X the lower limit ofquantification (LLOQ) was 0.1 ng/ml and for CEA CD3 TCB molecule B 0.44ng/ml.

A biphasic decline was observed with a half-life of 184±40 hours(non-compartmental analysis) for CEA CD3 TCB molecule B versus 32±11hours for CEA CD3 TCB molecule X. A clearance (CL) of 7±0.9 ml/d/kg wasdetected for CEA CD3 TCB molecule B and 25±6 ml/d/kg for CEA CD3 TCBmolecule X. Overall, CEA CD3 TCB molecule B showed IgG-like propertiesand displayed a longer half-life and a slower clearance in cynomolgusmonkeys as compared to CEA CD3 TCB molecule X.

TABLE 14 Summary of pharmacokinetic parameters of CEA CD3 TCB molecule Bin serum after a single intravenous (bolus) administration of 0.01 mg/kgto cynomolgus monkeys (N = 3) Half Life CL Cmax AUC_(INF) Vc ID (h)(ml/clay/kg) (ng/ml) (h*ng/ml) (ml/kg) Mean 184 7 257 33351 39 SD 40 0.918 4259 2 CV % 22 13 7 13 6

TABLE 15 Summary of pharmacokinetic parameters of CEA CD3 TCB molecule Xin serum after a single intravenous (bolus) administration of 0.01 mg/kgto cynomolgus monkeys (N = 5). Half Life CL Cmax AUC_(INF) Vc ID (h)(ml/clay/kg) (ng/ml) (h*ng/ml) (ml/kg) Mean 32 25 321 9991 31 SD 11 6 242133 2 CV % 34 23 7 21 7

The Phoenix v6.4 from Pharsight Ltd was used for PK assessment.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. A T cell activating bispecific antigen bindingmolecule comprising (a) a first antigen binding moiety whichspecifically binds to a first antigen; (b) a second antigen bindingmoiety which specifically binds to a second antigen; wherein the firstantigen is an activating T cell antigen and the second antigen is CEA,or the first antigen is CEA and the second antigen is an activating Tcell antigen; and wherein the antigen binding moiety which specificallybinds to CEA comprises a heavy chain variable region, particularly ahumanized heavy chain variable region, comprising the heavy chaincomplementarity determining region (HCDR) 1 of SEQ ID NO: 14, the HCDR 2of SEQ ID NO: 15 and the HCDR 3 of SEQ ID NO: 16, and a light chainvariable region, particularly a humanized light chain variable region,comprising the light chain complementarity determining region (LCDR) 1of SEQ ID NO: 17, the LCDR 2 of SEQ ID NO: 18 and the LCDR 3 of SEQ IDNO:
 19. 2. The T cell activating bispecific antigen binding moleculeaccording to claim 1, wherein the antigen binding moiety whichspecifically binds to CEA comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 22and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO:
 23. 3. The T cell activating bispecificantigen binding molecule according to claim 1 or 2, wherein the firstand/or the second antigen binding moiety is a Fab molecule.
 4. The Tcell activating bispecific antigen binding molecule according to any oneof claims 1-3, wherein the second antigen binding moiety is a Fabmolecule which specifically binds to a second antigen, and wherein thevariable domains VL and VH or the constant domains CL and CH1 of the Fablight chain and the Fab heavy chain are replaced by each other.
 5. The Tcell activating bispecific antigen binding molecule according to any oneof claims 1-4, wherein the first antigen is CEA and the second antigenis an activating T cell antigen.
 6. The T cell activating bispecificantigen binding molecule according to any one of claims 1-5, wherein theactivating T cell antigen is CD3, particularly CD3 epsilon.
 7. The Tcell activating bispecific antigen binding molecule according to any oneof claims 1-6, wherein the antigen binding moiety which specificallybinds to the activating T cell antigen comprises the heavy chaincomplementarity determining region (CDR) 1 of SEQ ID NO: 4, the heavychain CDR 2 of SEQ ID NO: 5, the heavy chain CDR 3 of SEQ ID NO: 6, thelight chain CDR 1 of SEQ ID NO: 8, the light chain CDR 2 of SEQ ID NO: 9and the light chain CDR 3 of SEQ ID NO:
 10. 8. The T cell activatingbispecific antigen binding molecule according to any one of claims 1-7,wherein the antigen binding moiety which specifically binds to theactivating T cell antigen comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 3and a light chain variable region comprising an amino acid sequence thatis at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO:
 7. 9. The T cell activating bispecificantigen binding molecule according to any one of claims 1-8, wherein thefirst antigen binding moiety under (a) is a first Fab molecule whichspecifically binds to a first antigen, the second antigen binding moietyunder (b) is a second Fab molecule which specifically binds to a secondantigen wherein the variable domains VL and VH of the Fab light chainand the Fab heavy chain are replaced by each other; and i) in theconstant domain CL of the first Fab molecule under a) the amino acid atposition 124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat), and wherein in theconstant domain CH1 of the first Fab molecule under a) the amino acid atposition 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index); or ii) in the constant domain CL of thesecond Fab molecule under b) the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat), and wherein in the constant domain CH1of the second Fab molecule under b) the amino acid at position 147 orthe amino acid at position 213 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index).10. The T cell activating bispecific antigen binding molecule accordingto claim 9, wherein in the constant domain CL of the first Fab moleculeunder a) the amino acid at position 124 is substituted independently bylysine (K), arginine (R) or histidine (H) (numbering according toKabat), and wherein in the constant domain CH1 of the first Fab moleculeunder a) the amino acid at position 147 or the amino acid at position213 is substituted independently by glutamic acid (E), or aspartic acid(D) (numbering according to Kabat EU index).
 11. The T cell activatingbispecific antigen binding molecule according to claim 9 or 10, whereinin the constant domain CL of the first Fab molecule under a) the aminoacid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat), andwherein in the constant domain CH1 of the first Fab molecule under a)the amino acid at position 147 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index).12. The T cell activating bispecific antigen binding molecule accordingto any one of claims 9-11, wherein in the constant domain CL of thefirst Fab molecule under a) the amino acid at position 124 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat) and the amino acid at position 123 issubstituted independently by lysine (K), arginine (R) or histidine (H)(numbering according to Kabat), and wherein in the constant domain CH1of the first Fab molecule under a) the amino acid at position 147 issubstituted independently by glutamic acid (E), or aspartic acid (D)(numbering according to Kabat EU index) and the amino acid at position213 is substituted independently by glutamic acid (E), or aspartic acid(D) (numbering according to Kabat EU index).
 13. The T cell activatingbispecific antigen binding molecule according to any one of claims 9-12,wherein in the constant domain CL of the first Fab molecule under a) theamino acid at position 124 is substituted by lysine (K) (numberingaccording to Kabat) and the amino acid at position 123 is substituted byarginine (R) (numbering according to Kabat), and wherein in the constantdomain CH1 of the first Fab molecule under a) the amino acid at position147 is substituted by glutamic acid (E) (numbering according to Kabat EUindex) and the amino acid at position 213 is substituted by glutamicacid (E) (numbering according to Kabat EU index).
 14. The T cellactivating bispecific antigen binding molecule according to any one ofclaims 9-12, wherein in the constant domain CL of the first Fab moleculeunder a) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) (numbering according to Kabat), and wherein inthe constant domain CH1 of the first Fab molecule under a) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).
 15. The T cell activating bispecific antigen binding moleculeaccording to claim 9, wherein in the constant domain CL of the secondFab molecule under b) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat), and wherein in the constant domain CH1 of thesecond Fab molecule under b) the amino acid at position 147 or the aminoacid at position 213 is substituted independently by glutamic acid (E),or aspartic acid (D) (numbering according to Kabat EU index).
 16. The Tcell activating bispecific antigen binding molecule according to claim 9or 15, wherein in the constant domain CL of the second Fab moleculeunder b) the amino acid at position 124 is substituted independently bylysine (K), arginine (R) or histidine (H) (numbering according toKabat), and wherein in the constant domain CH1 of the second Fabmolecule under b) the amino acid at position 147 is substitutedindependently by glutamic acid (E), or aspartic acid (D) (numberingaccording to Kabat EU index).
 17. The T cell activating bispecificantigen binding molecule according to any one of claim 9, and 16,wherein in the constant domain CL of the second Fab molecule under b)the amino acid at position 124 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat) andthe amino acid at position 123 is substituted independently by lysine(K), arginine (R) or histidine (H) (numbering according to Kabat), andwherein in the constant domain CH1 of the second Fab molecule under b)the amino acid at position 147 is substituted independently by glutamicacid (E), or aspartic acid (D) (numbering according to Kabat EU index)and the amino acid at position 213 is substituted independently byglutamic acid (E), or aspartic acid (D) (numbering according to Kabat EUindex).
 18. The T cell activating bispecific antigen binding moleculeaccording to any one of claims 9 and 15-17, wherein in the constantdomain CL of the second Fab molecule under b) the amino acid at position124 is substituted by lysine (K) (numbering according to Kabat) and theamino acid at position 123 is substituted by arginine (R) (numberingaccording to Kabat), and wherein in the constant domain CH1 of thesecond Fab molecule under b) the amino acid at position 147 issubstituted by glutamic acid (E) (numbering according to Kabat EU index)and the amino acid at position 213 is substituted by glutamic acid (E)(numbering according to Kabat EU index).
 19. The T cell activatingbispecific antigen binding molecule according to any one of claims 9 and15-17, wherein in the constant domain CL of the second Fab moleculeunder b) the amino acid at position 124 is substituted by lysine (K)(numbering according to Kabat) and the amino acid at position 123 issubstituted by lysine (K) (numbering according to Kabat), and wherein inthe constant domain CH1 of the second Fab molecule under b) the aminoacid at position 147 is substituted by glutamic acid (E) (numberingaccording to Kabat EU index) and the amino acid at position 213 issubstituted by glutamic acid (E) (numbering according to Kabat EUindex).
 20. The T cell activating bispecific antigen binding moleculeaccording to any one of claims 1-19, further comprising c) a thirdantigen binding moiety which specifically binds to the first antigen.21. The T cell activating bispecific antigen binding molecule accordingto claim 20, wherein the third antigen binding moiety is a Fab molecule.22. The T cell activating bispecific antigen binding molecule accordingto claim 20 or 21, wherein the third antigen binding moiety is identicalto the first antigen binding moiety.
 23. The T cell activatingbispecific antigen binding molecule according to any one of claims20-22, wherein the first and the third antigen binding moietyspecifically bind to a target cell antigen, and the second antigenbinding moiety specifically binds to an activating T cell antigen,particularly CD3, more particularly CD3 epsilon.
 24. The T cellactivating bispecific antigen binding molecule according to any one ofclaims 1 to 23, additionally comprising d) an Fc domain composed of afirst and a second subunit capable of stable association.
 25. The T cellactivating bispecific antigen binding molecule according to any one ofclaims 1 to 24, wherein the first and the second antigen binding moietyare fused to each other, optionally via a peptide linker.
 26. The T cellactivating bispecific antigen binding molecule according to any one ofclaims 1 to 25, wherein the first and the second antigen bindingmoieties are Fab molecules and the second antigen binding moiety isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the first antigen binding moiety.
 27. The T cellactivating bispecific antigen binding molecule of any one of claims 1 to25, wherein the first and the second antigen binding moieties are Fabmolecules and the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second antigen binding moiety.
 28. The T cell activatingbispecific antigen binding molecule of claim 26 or 27, wherein the firstand the second antigen binding moieties are Fab molecules and the Fablight chain of the first antigen binding moiety and the Fab light chainof the second antigen binding moiety are fused to each other, optionallyvia a peptide linker.
 29. The T cell activating bispecific antigenbinding molecule according to claim 24, wherein the first and the secondantigen binding moieties are Fab molecules and the second antigenbinding moiety is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first or the second subunit of the Fc domain.
 30. TheT cell activating bispecific antigen binding molecule according to claim24, wherein the first and the second antigen binding moieties are Fabmolecules and the first antigen binding moiety is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first or thesecond subunit of the Fc domain.
 31. The T cell activating bispecificantigen binding molecule according to claim 24, wherein the first andthe second antigen binding moieties are Fab molecules and the first andthe second antigen binding moiety are each fused at the C-terminus ofthe Fab heavy chain to the N-terminus of one of the subunits of the Fcdomain.
 32. The T cell activating bispecific antigen binding moleculeaccording to any one of claims 24, 29 or 30, wherein the third antigenbinding moiety is a Fab molecule and is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or second subunit of theFc domain.
 33. The T cell activating bispecific antigen binding moleculeof claim 24, wherein the first, second and third antigen bindingmoieties are Fab molecules and the second and the third antigen bindingmoiety are each fused at the C-terminus of the Fab heavy chain to theN-terminus of one of the subunits of the Fc domain, and the firstantigen binding moiety is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the second antigen bindingmoiety.
 34. The T cell activating bispecific antigen binding moleculeaccording to claim 24, wherein the first, second and third antigenbinding moieties are Fab molecules and the first and the third antigenbinding moiety are each fused at the C-terminus of the Fab heavy chainto the N-terminus of one of the subunits of the Fc domain, and thesecond antigen binding moiety is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding moiety.
 35. The T cell activating bispecific antigenbinding molecule according to claim 34, wherein the first and the thirdantigen binding moiety and the Fc domain are part of an immunoglobulinmolecule, particularly an IgG class immunoglobulin.
 36. The T cellactivating bispecific antigen binding molecule according to any one ofclaims 24-35, wherein the Fc domain is an IgG, specifically an IgG orIgG₄, Fc domain.
 37. The T cell activating bispecific antigen bindingmolecule according to any one of claims 24-36, wherein the Fc domain isa human Fc domain.
 38. The T cell activating bispecific antigen bindingmolecule according to any one of claims 24-37, wherein the Fc domaincomprises a modification promoting the association of the first and thesecond subunit of the Fc domain.
 39. The T cell activating bispecificantigen binding molecule of claim 38, wherein in the CH3 domain of thefirst subunit of the Fc domain an amino acid residue is replaced with anamino acid residue having a larger side chain volume, thereby generatinga protuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and in the CH3 domain of the second subunit of the Fc domain an aminoacid residue is replaced with an amino acid residue having a smallerside chain volume, thereby generating a cavity within the CH3 domain ofthe second subunit within which the protuberance within the CH3 domainof the first subunit is positionable.
 40. The T cell activatingbispecific antigen binding molecule of claim 39, wherein said amino acidresidue having a larger side chain volume is selected from the groupconsisting of arginine (R), phenylalanine (F), tyrosine (Y), andtryptophan (W), and said amino acid residue having a smaller side chainvolume is selected from the group consisting of alanine (A), serine (S),threonine (T), and valine (V).
 41. The T cell activating bispecificantigen binding molecule of claim 39 or 40, wherein in the CH3 domain ofthe first subunit of the Fc domain the threonine residue at position 366is replaced with a tryptophan residue (T366W), and in the CH3 domain ofthe second subunit of the Fc domain the tyrosine residue at position 407is replaced with a valine residue (Y407V), and optionally in the secondsubunit of the Fc domain additionally the threonine residue at position366 is replaced with a serine residue (T366S) and the leucine residue atposition 368 is replaced with an alanine residue (L368A) (numberingsaccording to Kabat EU index).
 42. The T cell activating bispecificantigen binding molecule of any one of claims 39-41, wherein in thefirst subunit of the Fc domain additionally the serine residue atposition 354 is replaced with a cysteine residue (S354C) or the glutamicacid residue at position 356 is replaced with a cysteine residue(E356C), and in the second subunit of the Fc domain additionally thetyrosine residue at position 349 is replaced by a cysteine residue(Y349C) (numberings according to Kabat EU index).
 43. The T cellactivating bispecific antigen binding molecule of any one of claims39-42, wherein the first subunit of the Fc domain comprises amino acidsubstitutions S354C and T366W, and the second subunit of the Fc domaincomprises amino acid substitutions Y349C, T366S, L368A and Y407V(numbering according to Kabat EU index).
 44. The T cell activatingbispecific antigen binding molecule according to any one of claims24-43, wherein the Fc domain exhibits reduced binding affinity to an Fcreceptor and/or reduced effector function, as compared to a native IgG₁Fc domain.
 45. The T cell activating bispecific antigen binding moleculeaccording to any one of claims 24-44, wherein the Fc domain comprisesone or more amino acid substitution that reduces binding to an Fcreceptor and/or effector function.
 46. The T cell activating bispecificantigen binding molecule according to claim 45, wherein said one or moreamino acid substitution is at one or more position selected from thegroup of L234, L235, and P329 (Kabat EU index numbering).
 47. The T cellactivating bispecific antigen binding molecule according to any one ofclaims 24-46, wherein each subunit of the Fc domain comprises threeamino acid substitutions that reduce binding to an activating Fcreceptor and/or effector function wherein said amino acid substitutionsare L234A, L235A and P329G (Kabat EU index numbering).
 48. The T cellactivating bispecific antigen binding molecule of any one of claims 44to 47, wherein the Fc receptor is an Fcγ receptor.
 49. The T cellactivating bispecific antigen binding molecule of any one of claims 44to 48, wherein the effector function is antibody-dependent cell-mediatedcytotoxicity (ADCC).
 50. One or more isolated polynucleotide encodingthe T cell activating bispecific antigen binding molecule of any one ofclaims 1 to
 49. 51. One or more vector, particularly expression vector,comprising the polynucleotide(s) of claim
 50. 52. A host cell comprisingthe polynucleotide(s) of claim 50 or the vector(s) of claim
 51. 53. Amethod of producing a T cell activating bispecific antigen bindingmolecule capable of specific binding to CEA and an activating T cellantigen, comprising the steps of a) culturing the host cell of claim 52under conditions suitable for the expression of the T cell activatingbispecific antigen binding molecule and b) optionally recovering the Tcell activating bispecific antigen binding molecule.
 54. A T cellactivating bispecific antigen binding molecule produced by the method ofclaim
 53. 55. A pharmaceutical composition comprising the T cellactivating bispecific antigen binding molecule of any one of claims 1 to49 or 54 and a pharmaceutically acceptable carrier.
 56. A method oftreating a disease in an individual, comprising administering to saidindividual a therapeutically effective amount of a compositioncomprising the T cell activating bispecific antigen binding molecule ofany one of claims 1 to 49 or 54 in a pharmaceutically acceptable form.57. The method of claim 56, wherein said disease is cancer.
 58. A methodfor inducing lysis of a target cell, comprising contacting a target cellwith the T cell activating bispecific antigen binding molecule of anyone of claims 1-49 or 54 in the presence of a T cell.